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D Labuda

Publications and source records attributed to D Labuda.

33 records · Page 2Linked to original sources
PubMed1989

Sequence conservation in Alu evolution.

A statistical analysis of a set of genomic human Alu elements is based on a published alignment and a recent classification of these sequences. After separation of the Alu sequences into families, the consensus sequences of these families are determined, using the correct weighting of the unidirectional decay of CG-dinucleotides. For, the tenfold greater mutation rate at CG's requires separate consideration of an independent clock at every stage of analysis. The distributions of the substitutions with respect to the new consensus sequences, taking the CG and the non-CG-nucleotide positions separately, lie far closer to the expected distributions than the total diversity. Computer analysis of the folding of RNAs derived from these sequences indicates that RNA secondary structure is conserved among Alu families, suggesting its importance for Alu proliferation and/or function. The folding pattern, further substantiated by a number of compensatory mutations, includes secondary structure domains which are homologous to those observed in 7SL RNA and a defined region of interaction between the two Alu subunits. These results are consistent with a model in which a small number of conserved Alu master genes give rise via retroposition to the numerous copies of Alu pseudogenes, that then diversify by random substitution. The master genes appeared at different periods during evolution giving rise to different families of Alu sequences.

Base Composition↗
PubMed1989

The gene for incontinentia pigmenti is assigned to Xq28.

A linkage study of eight families with incontinentia pigmenti (IP) has been performed, and linkage to site DXS52 has been established. We suggest that the IP locus lies in the Xq terminal region on the long arm of the X chromosome.

Chromosome Mapping↗
PubMed1989

The three conformations of the anticodon loop of yeast tRNA(Phe).

The complex conformational states of the anticodon loop of yeast tRNA(Phe) which we had previously studied with relaxation experiments by monitoring fluorescence of the naturally occurring Wye base, are analyzed using time and polarization resolved fluorescence measurements at varying counterion concentrations. Synchrotron radiation served as excitation for these experiments, which were analyzed using modulating functions and global methods. Three conformations of the anticodon loop are detected, all three occurring in a wide range of counterion concentrations with and without Mg2+, each being identified by its typical lifetime. The fluorescence changes brought about by varying the ion concentrations, previously monitored by steady state fluorimetry and relaxation methods, are changes in the population of these three conformational states, in the sense of an allosteric model, where the effectors are the three ions Mg2+, Na+ and H+. The population of the highly fluorescent M conformer (8ns), most affine to magnesium, is thus enhanced by that ligand, while the total fluorescence decreases as lower pH favors the H+-affine H conformer (0.6ns). Na+-binding of the N conformer (4ns) is responsible for complex fluorescence changes. By iterative simulation of this allosteric model the equilibrium and binding constants are determined. In turn, using these constants to simulate equilibrium fluorescence titrations reproduces the published results.

Anticodon↗
PubMed1988

Lesch-Nyhan syndrome: molecular investigation of three French Canadian families using a hypoxanthine-guanine phosphoribosyltransferase cDNA probe.

Using human hypoxanthine-guanine phosphoribosyltransferase (HPRT) cDNA and an anonymous probe 36B-2, we examined the segregation of restriction fragment length polymorphism (RFLP) alleles with the Lesch-Nyhan phenotype in three affected families. Two families were informative. Five carriers of the mutation in one family and two potential carriers in the second were heterozygous for either one or both polymorphisms allowing for prenatal diagnosis. Southern blot patterns in patients from these three families indicated the absence of major structural alterations in the defective gene. Northern analysis using HPRT cDNA as a probe revealed no hybridizing RNA in one patient, whereas normal size mRNA was expressed at a very low level in the second and at a level comparable to normal in the third. These data are consistent with heterogeneity of Lesch-Nyhan genetic lesions resulting from point mutations or small DNA deletions or rearrangements, which may affect transcription, stability, or integrity of the HPRT message.

Blotting, Northern↗
PubMed1985

Mechanism of codon recognition by transfer RNA studied with oligonucleotides larger than triplets.

The binding of yeast tRNAPhe to UUCA, UUCC, UUCCC, UUCUUCU, U4, U5, U6 and U7 was analysed by fluorescence temperature jump and equilibrium sedimentation measurements. In all cases the two observed relaxation processes can be assigned to alpha) an intramolecular conformation change of the anticodon loop and beta) preferential binding of the oligonucleotides to one of the anticodon conformations. The anticodon loop transition is associated with inner sphere complexation of Mg2+ and proceeds with rate constants of about 10(3) s-1. The rate constants of oligonucleotide binding are between 4 and 10 X 10(6) M-1s-1 and reflect an increase of the association rate with the number of binding sites compensated to some degree by electrostatic repulsion in the preequilibrium complex. Neither temperature jump nor equilibrium sedimentation experiments provided evidence for UUCA or UUCC induced tRNA dimerisation, although UUC binding leads to strong tRNA dimerisation under equivalent conditions. The results obtained for the longer oligonucleotides are similar. In the case of UUCUUCU with its two potential binding sites for tRNAPhe there was no evidence for the formation of 'ternary' complexes. Apparently tRNAPhe binds preferentially to the second UUC of this 'messenger' and forms additional contacts with residues on either side of the codon. Some evidence for the formation of ternary complexes is obtained for U6 and U7, although the extent of this reaction remains very small. Our results demonstrate that the mode of tRNA binding to a codon is strongly influenced by residues next to the codon. The formation of cooperative contacts between tRNA molecules at adjacent codons apparently requires support by a catalyst adjusting an appropriate conformation of messenger and tRNA molecules.

Anticodon↗
PubMed1985

Cooperativity in low-affinity Mg2+ binding to tRNA.

The Pb2+-catalyzed cleavage of tRNAPhe has been used to probe the effect of Na+ and Mg2+ binding to tRNA. Na+ is a noncompetitive inhibitor of the Pb2+-catalyzed cleavage. Millimolar Mg2+ is also a noncompetitive inhibitor. Analysis of the Mg2+ data show that at least two sites are involved in binding and that there is an interaction between the sites (cooperativity). Low-affinity Mg2+ binding is thus different from "weak" and "strong" Mg2+ binding to tRNA characterized previously. We postulate that the alterations induced by low-affinity Mg2+ binding in tRNA mimic to some extent those brought about in RNA by the interaction with a protein factor and that at appropriate [Mg2+] the whole structure of tRNA is able to respond in a concerted way to a signal from the environment such as aminoacylation or codon binding.

Binding Sites↗
PubMed1977

Dependence of tRNA structure in solution upon ionic condition of the solvent. Fluorescence studies of monovalent cation binding to tRNAPhe from barley embryos.

Dependence of barley phenylalanine tRNA (tRNAPhe) fluorescence intensity at 430 nm upon LiCl, NaCl, KCl, CsCl or NH4Cl concentration was measured in 0.01 M Tris-HCl, pH 7.5, 0.001 M Na2EDTA solutions. Increase of monovalent cation concentration in the solvent from 0 to 2 M induced about 3-fold fluorescence intensity enhancement. The fractional fluorescence change was used as a measure of bound ligand concentration. Fluorescence Scatchard plots revealed three classes of monovalent cation binding sites on the tRNA molecule: interacting (strong) and independent (weak and very weak) sites. Calculated from Scatchard plots binding constants (K), for strong and weak binding of monovalent cations (in the case of Na+ binding: Ks = 26 M-1 and Kw = 4.3 M-1 respectively) exhibit linear dependence upon ionic radius (r). Two limiting values obtained from the plot of K versus r: K(max) at r = 0 r(max) at K = 0, characterize additionally strong and weak monovalent cation binding sites (Ks(max) = 42 M-1 Kw(max) = 8.5 M-1, rs(max) = 0.23 nm and rw(max) = 0.22 nm). A model of the relationship between weak Mg2+ binding sites and monovalent cation binding sites as well as of monovalent cations binding to tRNA is proposed.

Cations, Monovalent↗
PubMed1976

Large-scale isolation of tRNA from barley embryos.

1. Large-scale isolation of tRNA from barley embryos is described, involving: phenol extraction, RNA deproteinization with the chloroform-isoamyl alcohol mixture, batch sorption on DEAE-cellulose, NaCl gradient elution of tRNA from DEAE-cellulose, and deaminoacylation of tRNA in the presence of bentonite. The procedure yielded tRNA free of protein and RNase activity. 2. The amino acid acceptor activity of the crude barley tRNA, its melting profiles and chromatographic patterns on Sephadex G-100 and BD-cellulose were similar to those of tRNA from other sources.

Amino Acyl-tRNA Synthetases↗