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D Gautheret

Publications and source records attributed to D Gautheret.

17 recordsLinked to original sources
PubMed1998

Quantitative analysis of the T cell repertoire selected by a single peptide-major histocompatibility complex.

The positive selection of CD4+ T cells requires the expression of major histocompatibility complex (MHC) class II molecules in the thymus, but the role of self-peptides complexed to class II molecules is still a matter of debate. Recently, it was observed that transgenic mice expressing a single peptide-MHC class II complex positively select significant numbers of diverse CD4+ T cells in the thymus. However, the number of selected T cell specificities has not been evaluated so far. Here, we have sequenced 700 junctional complementarity determining regions 3 (CDR3) from T cell receptors (TCRs) carrying Vbeta11-Jbeta1.1 or Vbeta12-Jbeta1.1 rearrangements. We found that a single peptide-MHC class II complex positively selects at least 10(5) different Vbeta rearrangements. Our data yield a first evaluation of the size of the T cell repertoire. In addition, they provide evidence that the single Ealpha52-68-I-Ab complex skews the amino acid frequency in the TCR CDR3 loop of positively selected T cells. A detailed analysis of CDR3 sequences indicates that a fraction of the beta chain repertoire bears the imprint of the selecting self-peptide.

Animals↗
PubMed1998

Alternate polyadenylation in human mRNAs: a large-scale analysis by EST clustering.

Alternate polyadenylation is an important post-transcriptional regulatory process now open to large-scale analysis by use of cDNA databases. We clustered 164,000 expressed sequence tags (ESTs) into approximately 15,000 groups and aligned each group to a putative mRNA 3' end. By use of stringent criteria to discard artifactual mRNA extremities, clear evidence for alternate polyadenylation was obtained in 189 of the 1000 EST clusters studied. A number of previously unreported polyadenylation sites were identified, together with possible instances of tissue-specific differential polyadenylation. This study demonstrates that, besides quantitative aspects of gene expression, the distribution of alternate mRNA forms can be analyzed through EST sampling.

Computational Biology↗
PubMed1997

No tRNA3Lys unwinding in a complex with HIV NCp7.

The nucleocapsid protein NCp7 of the human immunodeficiency virus (HIV) type 1 is important for the annealing of HIV RNA and tRNA3Lys, the tRNA acting as a primer during reverse transcription of HIV RNA. A wild type NCp7 and a Cys23 mutant having a disrupted zinc finger were analyzed with far UV circular dichroism (CD). CD data analysis revealed that NCp7 has a high content of extended structures in aqueous buffer, decreasing in Cys23 NCp7 and in NCp7 in the absence of zinc. An increase in beta-turn structures is observed in NCp7 bound to tRNA3Lys. Furthermore, CD data shows that Cys23 NCp7 binds tRNA3Lys. The CD spectrum of tRNA3Lys is typical of an A-form helix and retains this structure after binding of NCp7, which demonstrates that NCp7 does not induce tRNA3Lys unwinding. CD spectra of tRNA3Lys were measured from 5 to 80 degrees C to observe CD changes resulting from tRNA3Lys melting. Molecular modeling of the complex identifies two potential tRNA anticodon binding sites in the NCp7 N-terminal region and first zinc finger. In this model, both binding sites can interact with 12 nucleotides in the anticodon domain without requiring a base specificity.

Amino Acid Sequence↗
PubMed1997

Inferring the conformation of RNA base pairs and triples from patterns of sequence variation.

The success of comparative analysis in resolving RNA secondary structure and numerous tertiary interactions relies on the presence of base covariations. Although the majority of base covariations in aligned sequences is associated to Watson-Crick base pairs, many involve non-canonical or restricted base pair exchanges (e.g. only G:C/A:U), reflecting more specific structural constraints. We have developed a computer program that determines potential base pairing conformations for a given set of paired nucleotides in a sequence alignment. This program (ISOPAIR) assumes that the base pair conformation is maintained through sequence variation without significantly affecting the path of the sugar-phosphate backbone. ISOPAIR identifies such 'isomorphic' structures for any set of input base pair or base triple sequences. The program was applied to base pairs and triples with known structures and sequence exchanges. In several instances, isomorphic structures were correctly identified with ISOPAIR. Thus, ISOPAIR is useful when assessing non-canonical base pair conformations in comparative analysis. ISOPAIR applications are limited to those cases where unusual base pair exchanges indeed reflect a non-canonical conformation.

Base Composition↗
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

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

Fitting the structurally diverse animal mitochondrial tRNAs(Ser) to common three-dimensional constraints.

We propose three-dimensional models for animal mitochondrial (amt) tRNAs lacking the D-domain based on consideration of universal constraints on tRNA to maintain functionality. The available tRNA sequences are classified into two groups, and distinct models are proposed for both classes derived from common structural features. The distance between the anticodon and the acceptor stem is comparable in the models and corresponds to that observed in conventional tRNAs. This fact averts the problem of how a shorter mitochondrial tRNA could function within the context of a protein synthesis machinery suited to full-sized tRNAs. In the models, the angle which defines the relationship between the helical domains composed of the acceptor/T-stem and the anticodon/D-stem is greater than in conventional tRNAs. These structures resemble more a "boomerang" than an "L". However, even in the boomerang model, the inner surface of tRNA would be sufficiently uncluttered to avoid steric clashes when two tRNA molecules cohabit the ribosome.

Animals↗
PubMed1993

Reproducing the three-dimensional structure of a tRNA molecule from structural constraints.

The three-dimensional structure of yeast tRNA(Phe) was reproduced at atomic resolution with the automated RNA modeling program MC-SYM, which is based on a constraint-satisfaction algorithm. Structural constraints used in the modeling were derived from the secondary structure, four tertiary base pairs, and other information available prior to the determination of the x-ray crystal structure of the tRNA. The program generated 26 solutions (models), all of which had the familiar "L" form of tRNA and root-mean-square deviations from the crystal structure in the range of 3.1-3.8 A. The interaction between uridine-8 and adenosine-14 was crucial in the modeling procedure, since only this among the tertiary pairs is necessary and sufficient to reproduce the L form of tRNA. Other tertiary interactions were critical in reducing the number of solutions proposed by the program.

Base Sequence↗
PubMed1993

Modeling the three-dimensional structure of RNA using discrete nucleotide conformational sets.

The flexibility about seven torsion angles in nucleotides constitutes a severe obstacle to computer modeling of RNA. The computational feasibility of RNA conformational searches can be enhanced by assigning to each nucleotide a set of discrete conformations. In this work, four types of discrete conformational sets for the atomic representation of nucleotide structures were defined and evaluated. These sets, comprising between 10 and 30 conformations, were tested for their ability to reproduce known RNA structures and to generate structures responding to new specifications. Conformational searches were performed with the MC-SYM program, which allows for the generation of all structures satisfying a predetermined set of three-dimensional constraints in a given discrete space. Results with known hairpin loop structures show that root-mean-square deviations of about 1.5 A for backbone atoms and about 2.0 A for all atoms between the modeled and X-ray crystal structures can be expected. The conformational set that gives the most faithful representation of test structures is based on the classification of nucleotide conformations derived from a structural database. Representative conformations are selected from each class that adequately sample variations in backbone direction, sugar pucker and base orientation. With this conformational set, most of the important features of test hairpin structures are reproduced with fidelity, indicating that biologically useful models can be constructed from the combination of discrete nucleotide conformations and an algorithm that rapidly and systematically scans the pre-defined conformational space.

Anticodon↗
PubMed1993

Modeling the three-dimensional structure of RNA.

The limited number of RNA structures determined by X-ray crystallography and NMR spectroscopy compels the use of experimental and theoretical methods that are less precise to obtain information on RNA conformation. RNA flexibility, a consequence of rotational freedom about seven intra- and internucleotide bonds, is unfortunately of such magnitude that these alternate techniques fall short of providing sufficient information to build robust tertiary structures. Various RNA modeling methods, described herein, permit the organization of this structural data to the form of three-dimensional structures. Interactive computer graphics techniques, for example, have generated several useful models. Also, conventional computer algorithms involving the minimization of empirical energy functions, previously limited to small molecules, are giving way to methods able to handle much larger molecules. Modified distance geometry and molecular mechanics algorithms, using simplified "pseudoatom" representations, can generate structures consistent with input data. A constraint satisfaction algorithm combined with discrete representations of nucleotide conformations systematically explores poorly defined regions of a molecule yielding all-atom representations, but requires enough structural constraints to avoid a computational explosion.

Algorithms↗
PubMed1991

The combination of symbolic and numerical computation for three-dimensional modeling of RNA.

Three-dimensional (3-D) structural models of RNA are essential for understanding of the cellular roles played by RNA. Such models have been obtained by a technique based on a constraint satisfaction algorithm that allows for the facile incorporation of secondary and other structural information. The program generates 3-D structures of RNA with atomic-level resolution that can be refined by numerical techniques such as energy minimization. The precision of this technique was evaluated by comparing predicted transfer RNA loop and RNA pseudoknot structures with known or consensus structures. The root-mean-square deviation (2.0 to 3.0 angstroms before minimization) between predicted and control structures reveal this system to be an effective method in modeling RNA.

Algorithms↗
PubMed1990

Pattern searching/alignment with RNA primary and secondary structures: an effective descriptor for tRNA.

A convenient pattern-matching program using primary and higher-order structural features has been developed based on a 'backtracking' algorithm. A second implementation of the algorithm uses descriptors of structural features (including primary sequences) to align a list of homologous or highly similar sequences. An application of the pattern matcher to the search for tRNA and group I intron structural motifs in sequence data banks is presented. The design of a highly discriminate tRNA motif, common to all cellular tRNAs and not found in a control sequence bank, was accomplished using the pattern matcher in conjunction with the alignment program.

Algorithms↗
PubMed1988

A secondary and tertiary structure editor for nucleic acids.

A major difficulty in the evaluation of secondary and tertiary structures of nucleic acids is the lack of convenient methods for their construction and representation. As a first step in a study of the symbolic representation of biopolymers, we report the development of a structure editor written in Pascal, permitting model construction on the screen of a personal computer. The program calculates energies for helical regions, allows user-defined helices and displays the secondary structure of a nucleic acid based on a user-selected set of helices. Screen and printer outputs can be in the form of a backbone or the letters of the primary sequence. The molecule can then be displayed in a format which simulates its three-dimensional structure. Using appropriate glasses, the molecule can be viewed on the screen in three dimensions. Other options include the manipulation of helices and single-stranded regions which results in changes in the spatial relationship between different regions of the molecule. The editor requires an IBM or compatible PC, 640 kbyte memory and a medium or high resolution graphics card.

Algorithms↗