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D Labuda

Publications and source records attributed to D Labuda.

At least 73 records · Page 4Linked to original sources
PubMed1991

Relationship between 2'-hydroxyls and magnesium binding in the hammerhead RNA domain: a model for ribozyme catalysis.

The use of deoxyribonucleotide substitution in RNA (mixed RNA/DNA polymers) permits an evaluation of the role of 2'-hydroxyl groups in ribozyme catalysis. Specific deoxyribonucleotide substitution at G9 and A13 of the ribozyme decreases the catalytic activity (kcat) of the ribozyme by factors of 14 and 20, respectively. The reduction of the reaction rate concomitant with the absence of these 2'-OHs or the 2'-OH of the substrate U7 position can be partially compensated by increasing the Mg2+ concentration above 10 mM. The KMg of the all-RNA ribozyme is 5.3 mM, and the lack of either of the three influential 2'-OHs increases this value by a factor of approximately 3. These and other reaction constants for the ribozyme and the deoxy-substituted analogues have been determined by assuming a three-step mechanism. The data presented here provide the basis for the formulation of a molecular model of ribozyme activity.

Base Sequence↗
PubMed1991

Splice-mediated insertion of an Alu sequence inactivates ornithine delta-aminotransferase: a role for Alu elements in human mutation.

In studies of mutations causing deficiency of ornithine delta-aminotransferase (EC 2.6.1.13), we found an allele whose mature mRNA has a 142-nucleotide insertion at the junction of sequences from exons 3 and 4. The insert derives from an Alu element in ornithine delta-aminotransferase intron 3 oriented in the direction opposite to transcription (an "antisense Alu"). A guanine----cytosine transversion creates a donor splice site in this Alu, activating a cryptic acceptor splice site at its 5' end and causing splice-mediated insertion of an Alu fragment into the mature ornithine-delta-aminotransferase mRNA. We note that the complement of the Alu consensus sequence has at least two cryptic acceptor sites and several potential donor sequences and predict that similar mutations will be found in other genes.

Amino Acid Sequence↗
PubMed1991

Evolution of mouse B1 repeats: 7SL RNA folding pattern conserved.

In a recent report mouse B1 genomic repeats were divided into six families representing different waves of fixation of B1 variants, consistent with the retroposition model of human Alu elements. These data are used to examine the distribution of nucleotide substitutions in individual genomic repeats with respect to family consensus sequences and to compare the minimal energy structures of the corresponding B1 RNAs. By an enzymatic approach the predicted structure of B1 RNAs is experimentally confirmed using as a model sequence an RNA of a young B1 family member transcribed in vitro by T7 RNA polymerase. B1 RNA preserves folding domains of the Alu fragment of 7SL RNA, its progenitor molecule. Our results reveal similarities among 7SL-like retroposons, human Alu, and rodent B1 repeats, and relate the evolutionary conservation of B1 family consensus sequences to selection at the RNA level.

Animals↗
PubMed1991

G proteins in normal rat pituitaries and in prolactin-secreting rat pituitary tumors.

It is still undetermined which GTP-binding (G) protein is involved in the regulation of prolactin (PRL) release and through which effector. This study shows that, when compared to normal pituitary tissue, the levels of alpha o protein were very low in dopamine (DA)-resistant, PRL-secreting pituitary tumors 7315a and MtTW15, while alpha o mRNA was present in the two tumors. In the MtTW15 tumor alpha i1, alpha i2 and alpha i3 levels were decreased while those of alpha s42 and alpha s47 were increased, and in the 7315a tumor alpha i2, alpha i3 and beta levels were decreased and those of alpha s47 increased. In an estrone-induced, DA-sensitive prolactinoma the levels of alpha i3 were greatly reduced. DA was unable to inhibit basal PRL release by 7315a and MtTW15 and basal cAMP accumulation by adenomatous and MtTW15 cells. Vasoactive intestinal peptide (VIP) increased both cAMP accumulation and PRL release by all cell preparations which could be suppressed by DA with adenomatous and 7315a but not with MtTW15 cells. These and previously published results provide circumstantial evidence that alpha o, alpha i1 and alpha i3 are all involved in the transduction of the DA inhibitory message while alpha s47 transduces cAMP activating messages and alpha s42 is responsible for the constitutive activation of L-type Ca2+ channels, adenylate cyclase and baseline PRL release.

Adenoma↗
PubMed1990

Mixed DNA/RNA polymers are cleaved by the hammerhead ribozyme.

A series of chemically synthesized oligodeoxyribonucleotides containing one or two ribonucleotides (DNA/RNA mixed polymers) at and/or adjacent to the cleavage site of the substrate can be cleaved by the "hammerhead" ribozyme. In comparison with the all-RNA substrate, the predominantly deoxyribonucleotide substrates have (1) lower optimal temperatures of cleavage, (2) approximately 6-fold higher Km's and 7-fold lower kcat's at 30 degrees C, and (3) 15-fold higher Km's and 8-fold lower kcat's at 37 degrees C. The extent to which the RNA substrate cleavage is inhibited in the presence of an all-DNA (KI = 13 microM) and an RNA substrate analogue with a dC at the cleavage site (KI = 0.96 microM) supports the contention that the formation of the ribozyme-substrate complex with the predominantly deoxyribonucleotide substrates (D substrates) is impaired. The weaker binding of D substrates was confirmed by thermal denaturation and determination of the Tm of the complex. Analysis of the kinetic data also suggests that the conformation of the catalytic core of the ribozyme-substrate complex differs from that of the all-RNA complex, a change that results from the presence of a DNA/RNA heteroduplex in the complex.

Base Sequence↗
PubMed1990

Reverse transcriptase activity from human embryonal carcinoma cells NTera2D1.

We have identified an RNA-dependent DNA polymerase activity in the microsomal fraction of human pluri-potential embryonal carcinoma cells NTera2D1, which are known to express the full length coding strand of the genomic Line-1 (L1) elements. This activity was classified as a reverse transcriptase (RT) based on its utilization of an RT specific synthetic poly(Cm) template in the presence of Mn2+ ions. Treatment of the cell by ultraviolet irradiation (200 erg/mm2) which resulted in a 2- to 3-fold enhancement of the RT activity, was required for the reproducible detection of the activity throughout the entire purification procedure. More than a 100-fold enrichment in RT activity was obtained by centrifugation in a glycerol step gradient and a linear sucrose density gradient followed by Sephacryl S-1000 gel filtration. These experiments demonstrated that the RT activity was associated with a macromolecular complex having the characteristics of a viral-like particle with a major protein component of 37 kd. The presence of L1 mRNA in RT-containing fractions suggests that the activity identified could originate from L1 elements and/or be involved in the mechanism of retroposition.

Base Sequence↗
PubMed1990

Alumorphs--human DNA polymorphisms detected by polymerase chain reaction using Alu-specific primers.

The simultaneous analysis of multiple loci could substantially increase the efficiency of mapping studies. Toward this goal, we used the polymerase chain reaction to amplify multiple DNA fragments originating from dispersed genomic segments that are flanked by Alu repeats. Analysis of different human DNA samples revealed numerous amplification products distinguishable by size, some of which vary between individuals. A family study demonstrated that these polymorphic fragments are inherited in a Mendelian fashion. Because of the ubiquitous distribution of Alu repeats, these markers, called "alumorphs," could be useful for linkage mapping of the human genome. A major advantage of alumorphs is that no prior knowledge of DNA sequence of marker loci is required. This approach may find general application for any genome where interspersed repetitive sequences are found.

Base Sequence↗
PubMed1990

Leber's congenital amaurosis with Duchenne's muscular dystrophy.

We studied a family with Leber's congenital amaurosis and Duchenne's muscular dystrophy. One son, the proband, was noted to be blind from shortly after birth and had absent pupillary reflexes, attenuation of retinal blood vessels and pigmentary changes of the retina. He manifested Duchenne's muscular dystrophy at 6 years of age and died of its complication--severe respiratory failure--at 21 years of age. His older brother also was blind since birth, with absent pupillary light reflexes and an extinguished electroretinogram in both eyes. The proband's younger brother with Duchenne's muscular dystrophy died following respiratory failure. Their oldest brother was apparently unaffected. Their mother and sister were proven carriers of Duchenne's muscular dystrophy and a maternal uncle and great-uncle also died from this condition. The parents were consanguineous, third generation cousins.

Adolescent↗
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↗