Biomedical subjects

R R Gutell

Publications and source records attributed to R R Gutell.

At least 37 records · Page 2Linked to original sources

PubMed1997

Mutagenesis and comparative sequence analysis of a base triple joining the two domains of group I ribozymes.

Tertiary interactions are important in the higher-order folding of catalytic RNAs. Recently, a base triple, joining the two major domains of the catalytic core, was determined in group I introns from the cyanobacterium Anabaena PCC7120 and the eukaryote Tetrahymena thermophila. This base triple involves the fifth base pair of P4 and the fifth base of the single-stranded region J8/7. We made base pair and single-nucleotide substitutions in the fifth base pair of P4, a G-C in the wild-type Anabaena intron, and tested them for self-splicing activity. The results suggest a hydrogen bonding model in which only the C of the base pair interacts directly with the fifth base of J8/7. Comparative sequence analysis was used to determine the different combinations of base triples that occur in approximately 450 natural group I introns identified to date. About 94% of the base triples analyzed are compatible with the proposed hydrogen bonding model. Disrupting this base triple in the Tetrahymena intron resulted in the disappearance of splicing intermediates (intron 3' exon and 5' exon), even though the first step of splicing was not affected. Restoration of the base triple by a compensatory mutation reverted the intermediates to wild-type levels. These results suggest that disruption of the base triple increases the rate of the second step of splicing or of a conformational change preceding the second step. Repositioning of the base triple to form a new set of interactions may be required for the second step of splicing.

Anabaena↗

PubMed1996

A novel clade of protistan parasites near the animal-fungal divergence.

Sequences of nuclear-encoded small-subunit rRNA genes have been determined for representatives of the enigmatic genera Dermocystidium, Ichthyophonus, and Psorospermium, protistan parasites of fish and crustaceans. The small-subunit rRNA genes from these parasites and from the "rosette agent" (also a parasite of fish) together form a novel, statistically supported clade. Phylogenetic analyses demonstrate this clade to diverge near the animal-fungal dichotomy, although more precise resolution is problematic. In the most parsimonious and maximally likely phylogenetic frameworks inferred from the most stably aligned sequence regions, the clade constitutes the most basal branch of the metazoa; but within a limited range of model parameters, and in some analyses that incorporate less well-aligned sequence regions, an alternative topology in which it diverges immediately before the animal-fungal dichotomy was recovered. Mitochondrial cristae of Dermocystidium spp. are flat, whereas those of Ichthyophonus hoferi appear tubulovesiculate. These results extend our understanding of the types of organisms from which metazoa and fungi may have evolved.

Animals↗

PubMed1996

Characterization of a flatworm ribosomal RNA-encoding gene: promoter sequence and small subunit rRNA secondary structure.

The transcription start point (tsp) of a ribosomal RNA (rRNA)-encoding gene (rDNA) from Echinococcus granulosus has been mapped at a position located 1.1 kb upstream from the small subunit (SSU) rRNA coding sequence. As expected from the analysis of the putative promoter sequence (-200 to +50), no homology was found with rDNA promoters from other organisms. Nevertheless, some interesting motifs were found. There is a d(T)11 track flanked by an inverted repeat (IR) centered at position -32, which is analogous to the position of the TATA box in promoters transcribed by RNA polymerase II. Two other IR were observed that are also present in the Trypanosoma cruzi rDNA promoter. We have also determined the SSU rDNA sequence and established a secondary structure model. The analysis of the secondary structure strongly suggests that this gene encodes a functional SSU rRNA. The fact that both the promoter and the rRNA coding sequence are derived from the same rDNA repeat indicates that the promoter is also functional.

Animals↗

PubMed1996

Comprehensive comparison of structural characteristics in eukaryotic cytoplasmic large subunit (23 S-like) ribosomal RNA.

Comparative modeling of secondary structure is a proven approach to predicting higher order structural elements in homologous RNA molecules. Here we present the results of a comprehensive comparison of newly modeled or refined secondary structures for the cytoplasmic large subunit (23 S-like) rRNA of eukaryotes. This analysis, which covers a broad phylogenetic spectrum within the eukaryotic lineage, has defined regions that differ widely in their degree of structural conservation, ranging from a core of primary sequence and secondary structure that is virtually invariant, to highly variable regions. New comparative information allows us to propose structures for many of the variable regions that had not been modeled before, and rigorously to confirm or refine variable region structures previously proposed by us or others. The present analysis also serves to identify phylogenetically informative features of primary and secondary structure that characterize these models of eukaryotic cytoplasmic 23 S-like rRNA. Finally, the work summarized here provides a basis for experimental studies designed both to test further the validity of the proposed secondary structures and to explore structure-function relationships.

Base Sequence↗

PubMed1996

Phylogeny of the Chlamydomonadales (Chlorophyceae): a comparison of ribosomal RNA gene sequences from the nucleus and the chloroplast.

Phylogenetic analyses of nuclear-encoded small-subunit rRNA sequences and chloroplast-encoded large-subunit rRNA sequences from flagellate green algae representing the order Chlamydomonadales were found to show considerable congruence. In general, the chloroplast data set exhibited more robust support for comparable lineages than the nuclear data set. The phylogenetic inferences derived from the independent data sets support some, but also challenge many, traditional taxonomic and phylogenetic concepts regarding the green flagellates. Results from phylogenetic analyses of both molecular data sets support six distinct lineages that include taxa from the biflagellate genus, Chlamydomonas, and a basal lineage that comprises taxa from the quadriflagellate genus, Carteria. Both data sets support the conclusion that Chlamydomonas is not monophyletic. Although the chloroplast data are ambiguous regarding the question of Carteria monophyly, the nuclear data fail to support Carteria monophyly. The chlorococcalean genus Chlorococcum was found to have affinities with the Chlamydomonadales, indicating that the traditional concepts of both Chlorococcales and Chlamydomonadales may need revision. The genus Dunaliella is allied within the Chlamydomonadales, supporting the contention that it has lost a typical glycoprotein cell wall.

Animals↗

PubMed1996

An analysis of large rRNA sequences folded by a thermodynamic method.

BACKGROUND: The secondary structure of RNA can be predicted by the thermodynamics-based method of Zuker and Turner. The accuracy of the method's secondary structure predictions for rRNA can be assessed by using as reference the currently available rRNA secondary structure models that have been derived from comparative analysis of rRNA sequence alignments. RESULTS: We folded 72 23S rRNA sequences with the Zuker-Turner method and scored the resulting secondary structure predictions against the comparative model. Empirically, trends in the score were observed as a function of the phylogenetic memberships of the sequences and as a function of the base pairs secondary structural contexts. Further, three parameters were found that (anti-)correlate with the score. CONCLUSIONS: Three semiquantitative predictors of score were found: % of noncanonical base pairs, % of hairpin loops that were stable tetraloops, and sequence %G + C. The folding of rRNA is a tractable problem and thermodynamics-based folding algorithms, in particular, are useful in the study of this folding problem even for large RNA molecules (e.g. 16S and 23S rRNA).

Animals↗

PubMed1996

Structural features of the large subunit rRNA expressed in Plasmodium falciparum sporozoites that distinguish it from the asexually expressed subunit rRNA.

The developmentally regulated transcription of at least two distinct sets of nuclear-encoded ribosomal RNAs is detected in Plasmodium species. The identification of functional differences between the two sets of rRNAs is of interest. To facilitate the search for such differences, we have identified the 5.8S and 28S rRNAs from Plasmodium falciparum that are expressed in the sporozoite stage (S gene) of the parasites' life cycle in the mosquito host and compare them to transcripts expressed in the red blood cells (A gene) of the vertebrate host. This completes the first set of A- and S-type nuclear-encoded rRNA genes for a Plasmodium species. Analysis of the predicted secondary structures of the two units reveals the majority of differences between the A- and S-type genes occur in regions previously known to be variable. However, the predicted secondary structure of both 28S rRNAs indicates 11 positions within conserved areas that are not typical of eucaryotic rRNAs. Although the A-type gene resembles almost all eucaryotes, being atypical in only 4 of the 11 positions, the S gene is variant in 8 of the 11 positions. In three of these positions, the S-type gene resembles the consensus nucleotides for the 23S rRNA from Eubacteria and/or Archaea. A few differences occur in regions associated with ribosome function, in particular the GTPase site where the S-type differs in a base pair and loop from all known sequences. Further, the identification of compensatory changes at conserved points of interactions between the 5.8S-28S rRNAs indicates that transcripts from A- and S-units should not be interchangeable.

Animals↗

PubMed1995

Genetic and comparative analyses reveal an alternative secondary structure in the region of nt 912 of Escherichia coli 16S rRNA.

Mutations at position 912 of Escherichia coli 16S rRNA result in two notable phenotypes. The C-->U transition confers resistance to streptomycin, a translational-error-inducing antibiotic, while a C-->G transversion causes marked retardation of cell growth rate. Starting with the slow-growing G912 mutant, random mutagenesis was used to isolate a second site mutation that restored growth nearly to the wild-type rate. The second site mutation was identified as a G-->C transversion at position 885 in 16S rRNA. Cells containing the G912 mutation had an increased doubling time, abnormal sucrose gradient ribosome/subunit profile, increased sensitivity to spectinomycin, dependence upon streptomycin for growth in the presence of spectinomycin, and slower translation rate, whereas cells with the G912/C885 double mutation were similar to wild type in these assays. Comparative analysis showed there was significant covariation between positions 912 and 885. Thus the second-site suppressor analysis, the functional assays, and the comparative data suggest that the interaction between nt 912 and nt 885 is conserved and necessary for normal ribosome function. Furthermore, the comparative data suggest that the interaction extends to include G885-G886-G887 pairing with C912-U911-C910. An alternative secondary structure element for the central domain of 16S rRNA is proposed.

Drug Resistance, Microbial↗

PubMed1995

Complete sequences and organization of the rrnA operon from campylobacter jejuni TGH9011 (ATCC43431).

The rrnA ribosomal RNA (rRNA) operon of Campylobacter jejuni (Cj) TGH9011 (ATCC43431) was cloned and sequenced to completion. rRNAs were then characterized by primer extension and S1 nuclease mapping analysis. The secondary structure models of Cj 16S and 23S rRNAs were constructed, and the models were compared to the corresponding models from other eubacterial rRNA. The analysis presented a typical 5'-promoter-16S-tRNAs-23S-5S-terminator-3' prokaryotic rRNA operon structure. However, an unusual organization of the intercistronic tRNAs was observed where the two tRNAs, tRNA(Ala) and tRNA(Ile), were present in the order 5'-16S-tRNA(Ala)-tRNA(Ile)-23S-3', which is opposite of the typical 5'-16S-tRNA(Ile)-tRNA(Ala)-23S-3' structure observed in other bacteria.

Base Sequence↗

PubMed1995

Identification of base-triples in RNA using comparative sequence analysis.

Comparative sequence analysis has proven to be a very efficient tool for the determination of RNA secondary structure and certain tertiary interactions. However, base-triples, an important RNA structural element, cannot be predicted accurately from sequence data. We show here that the poor base correlations observed at base-triple positions are the result of two factors. (1) Base covariation is not as strictly required in triples as it is in Watson-Crick pairs. (2) Base-triple structures are less conserved among homologous molecules. A particularity of known triple-helical regions is the presence of multiple base correlations that do not reflect direct pairing. We suggest that natural mutations in base-triples create structural changes that require compensatory mutations in adjacent base-pairs and triples to maintain the triple-helix conformation. On the basis of these observations, we devised two new measures of association that significantly enhance the base-triple signal in correlation studies. We evaluated correlations between base-pairs and single stranded bases, and correlations between adjacent base-pairs. Positions that score well in both analyses are the best triple candidates. This procedure correctly identifies triples, or interactions very close to the proposed triples, in type I and type II tRNAs and in the group I intron.

Base Composition↗

PubMed1995

A comparison of thermodynamic foldings with comparatively derived structures of 16S and 16S-like rRNAs.

To increase our understanding of the dynamics and complexities of the RNA folding process, and therewith to improve our ability to predict RNA secondary structure by computational means, we have examined the foldings of a large number of phylogenetically and structurally diverse 16S and 16S-like rRNAs and compared these results with their comparatively derived secondary structures. Our initial goals are to establish the range of prediction success for this class of rRNAs, and to begin comparing and contrasting the foldings of these RNAs. We focus here on structural features that are predicted with confidence as well as those that are poorly predicted. Whereas the large set of Archaeal and (eu)Bacterial 16S rRNAs all fold well (69% and 55% respectively), some as high as 80%, many Eucarya and mitochondrial 16S rRNAs are poorly predicted (approximately 30%), with a few of these predicted as low as 10-20%. In general, base pairs interacting over a short distance and, in particular, those closing hairpin loops, are predicted significantly better than long-range base pairs and those closing multistem loops and bulges. The prediction success of hairpin loops varies, however, with their size and context. Analysis of some of the RNAs that do not fold well suggests that the composition of some hairpin loops (e.g., tetraloops) and the higher frequency of noncanonical pairs in their comparatively derived structures might contribute to these lower success rates. Eucarya and mitochondrial rRNAs reveal further novel tetraloop motifs, URRG/A and CRRG, that interchange with known stable tetraloop in the procaryotes.

Algorithms↗

PubMed1995

G.U base pairing motifs in ribosomal RNA.

An increasing number of recognition mechanisms in RNA are found to involve G.U base pairs. In order to detect new functional sites of this type, we exhaustively analyzed the sequence alignments and secondary structures of eubacterial and chloroplast 16S and 23S rRNA, seeking positions with high levels of G.U pairs. Approximately 120 such sites were identified and classified according to their secondary structure and sequence environment. Overall biases in the distribution of G.U pairs are consistent with previously proposed structural rules: the side of the wobble pair that is subject to a loss of stacking is preferentially exposed to a secondary structure loop, where stacking is not as essential as in helical regions. However, multiple sites violate these rules and display highly conserved G.U pairs in orientations that could cause severe stacking problems. In addition, three motifs displaying a conserved G.U pair in a specific sequence/structure environment occur at an unusually high frequency. These motifs, of which two had not been reported before, involve sequences 5'UG3' 3'GA5' and 5'UG3' 3'GU5', as well as G.U pairs flanked by a bulge loop 3' of U. The possible structures and functions of these recurrent motifs are discussed.

Base Composition↗

PubMed1994

A major family of motifs involving G.A mismatches in ribosomal RNA.

G.A oppositions and their flanking nucleotides in the internal loops of 16 S and 23 S rRNA were analyzed from a comparative structure perspective, resulting in an unexpectedly high incidence of the sequence motifs [formula: see text], [formula: see text] and [formula: see text]. The first two motifs can form similar three-dimensional structures containing sheared G.A or A.A pair conformations. Comparative sequence analysis revealed numerous sites in ribosomal RNAs with distinct combinations of nucleotides capable of forming this specific structure. In some cases, the sequence variations provide strong evidence for the sheared tandem structure occurring. Interestingly, the sequence changes would maintain a similar exposure of two adenines in the minor groove, suggesting the possibility that they are serving as a recognition or anchoring unit. These tandem conformations are related to that of the [formula: see text] tandem observed in 5 S and 28 S rRNA.

Hydrogen Bonding↗

PubMed1994

A molecular phylogeny of the marine red algae (Rhodophyta) based on the nuclear small-subunit rRNA gene.

A phylogeny of marine Rhodophyta has been inferred by a number of methods from nucleotide sequences of nuclear genes encoding small subunit rRNA from 39 species in 15 orders. Sequence divergences are relatively large, especially among bangiophytes and even among congeners in this group. Subclass Bangiophycidae appears polyphyletic, encompassing at least three lineages, with Porphyridiales distributed between two of these. Subclass Florideophycidae is monophyletic, with Hildenbrandiales, Corallinales, Ahnfeltiales, and a close association of Nemaliales, Acrochaetiales, and Palmariales forming the four deepest branches. Cermiales may represent a convergence of vegetative and reproductive morphologies, as family Ceramiaceae is at best weakly related to the rest of the order, and one of its members appears to be allied to Gelidiales. Except for Gigartinales, for which more data are required, the other florideophyte orders appear distinct and taxonomically justified. A good correlation was observed with taxonomy based on pit-plug ultrastructure. Tests under maximum-likelihood and parsimony of alternative phylogenies based on structure and chemistry refuted suggestions that Acrochaetiales is the most primitive florideophyte order and that Gelidiales and Hildenbrandiales are sister groups.

Base Sequence↗

PubMed1994

Partial nucleotide sequence of a single ribosomal RNA coding region and secondary structure of the large subunit 25 s rRNA of Candida albicans.

A rDNA cistron of Candida albicans strain WO-1 was cloned and the ITS1, ITS2, 5.8 s rDNA and 25 s rDNA coding regions sequenced in their entirety. These sequences were compared to those of three related yeast species (Saccharomyces cerevisiae, Saccharomyces carlsbergensis, and Thermomyces lanuginosus), and the 5.8 s rDNA was compared to seven additional 5.8 s rDNAs from organisms ranging in complexity from D. discoideum to H. sapiens. The C. albicans ITS regions are shorter than those of most other eukaryotes. The 25 s and 5.8 s rDNA sequences were folded into a secondary structure model based on comparative methods. In a comparison of regional similarities between the large subunit rDNAs of C. albicans, the three related yeasts and other eukaryotes, it is demonstrated that the additional sequences not present in the E. coli 23 s rDNA are more variable than the regions present in both prokaryotes and eukaryotes.

Animals↗

PubMed1994

Representation of the secondary and tertiary structure of group I introns.

Group I introns, which are widespread in nature, carry out RNA self-splicing. The secondary structure common to these introns was for the most part established a decade ago. Information about their higher order structure has been derived from a range of experimental approaches, comparative sequence analysis, and molecular modelling. This information now provides the basis for a new two-dimensional structural diagram that more accurately represents the domain organization and orientation of helices within the intron, the coaxial stacking of certain helices, and the proximity of key nucleotides in three-dimensional space. It is hoped that this format will facilitate the detailed comparison of group I intron structures.

Anabaena↗

PubMed1994

The 16S-like, 5.8S and 23S-like rRNAs of the two varieties of Cryptococcus neoformans: sequence, secondary structure, phylogenetic analysis and restriction fragment polymorphisms.

The nucleotide sequences of the 16S-like, 5.8S and 23S-like rDNAs from the two varieties of Cryptococcus neoformans, C. neoformans var. neoformans and C. neoformans var. gattii, were determined. The rRNA locus has the typical eukaryote organization of 16S-5.8S-23S with the 16S-like and 5.8S rRNA genes separated by a 124-nucleotide spacer and the 5.8S and 23S-like rRNA genes separated by a 187-nucleotide spacer in each strain. The C. neoformans var. neoformans and C. neoformans var. gattii 16S-like, 5.8S and 23S-like rRNAs are, respectively 1802, 158, and 3358 nucleotides in length and share > 99% nucleotide sequence identity, a finding which strongly supports the present taxonomic classification of two varieties within a species. Comparative structure analysis was used to construct secondary-structure models for the deduced 16S-like and 5.8S-23S-like rRNA sequences, which are similar to those of other fungal rRNAs. The C. neoformans 16S-like and 23S-like rRNA sequences were aligned with other eukaryote sequences based on secondary and higher-order structures predicted by comparative structure analysis for phylogenetic analysis. There was good correspondence between the 16S-like and 23S-like derived phylogenetic trees. The closest known fungal relative is Trichosporon beigelii. Southern blot analysis revealed one C. neoformans strain with two types of DNA repeats coding for rRNA which differed in size by about 1000 bp. Restriction fragment length polymorphisms in the rDNA locus provide useful markers for the study of epidemiology and pathogenesis of C. neoformans infections.

Base Sequence↗

PubMed1994

Collection of small subunit (16S- and 16S-like) ribosomal RNA structures: 1994.

A collection of diverse 16S and 16S-like rRNA secondary structure diagrams are available. This set of rRNAs contains representative structures from all of the major phylogenetic groupings--Archaea, (eu)Bacteria, and the nucleus, mitochondrion, and chloroplast of Eucarya. Within this broad phylogenetic sampling are examples of the major forms of structural diversity currently known for this class of rRNAs. These structure diagrams are available online through our computer-network WWW server and anonymous ftp, as well as from the author in hardcopy format.

Animals↗