Biomedical subjects

N Lumelsky

Publications and source records attributed to N Lumelsky.

5 recordsLinked to original sources

PubMed1989

Specific interactions in RNA enzyme-substrate complexes.

Analysis of crosslinked complexes of M1 RNA, the catalytic RNA subunit of ribonuclease P from Escherichia coli, and transfer RNA precursor substrates has led to the identification of regions in the enzyme and in the substrate that are in close physical proximity to each other. The nucleotide in M1 RNA, residue C92, which participates in a crosslink with the substrate was deleted and the resulting mutant M1 RNA was shown to cleave substrates lacking the 3' terminal CCAUCA sequence at sites several nucleotides away from the normal site of cleavage. The presence or absence of the 3' terminal CCAUCA sequence in transfer RNA precursor substrates markedly affects the way in which these substrates interact with the catalytic RNA in the enzyme-substrate complex. The contacts between wild-type M1 RNA and its substrate are in a region that resembles part of the transfer RNA "E" (exit) site in 23S ribosomal RNA. These data demonstrate that in RNA's with very different cellular functions, there are domains with similar structural and functional properties and that there is a nucleotide in M1 RNA that affects the site of cleavage by the enzyme.

Base Sequence↗

PubMed1989

Catalysis by the RNA subunit of RNase P--a minireview.

RNase P, an enzyme that contains both RNA and protein components, cleaves tRNA precursors to generate mature 5' termini. The catalytic activity of RNase P resides in the RNA component, with the protein cofactor affecting the rate of the cleavage reaction. The reaction is also influenced by the nature of the tRNA substrate.

Catalysis↗

PubMed1988

Selection and characterization of randomly produced mutants in the gene coding for M1 RNA.

The gene for M1 RNA, the catalytic subunit of RNase P of Escherichia coli, was subjected to random chemical mutagenesis in vitro. Mutations were selected by electrophoresis in denaturing gradient gels. Twenty-seven different mutants of the gene for M1 RNA were selected, and in 24 cases the mutations were identified as single base substitutions. The mutant forms of M1 RNA were analyzed in vitro for catalytic activity in the absence and in the presence of the protein subunit of RNase P (C5 protein). The structure of mutant RNAs was probed by limited digestion with ribonuclease T1; a correlation between reduced catalytic activity of mutant M1 RNAs and perturbations in secondary and tertiary structure was noted in many cases. The results indicate the involvement of specific regions of the M1 RNA molecule in the catalytic function of RNase P, in the binding of the C5 protein, and in substrate binding.

Endoribonucleases↗

PubMed1984

Sequence-determined DNA separations.

The variation in electrophoretic mobility of DNA under conditions of marginal helix stability provides a useful means for investigation of the relation between the helix-random chain transition and base sequence in natural DNA and a powerful procedure for separation of DNA molecules according to sequence. The use of statistical mechanical theory for analysis of the transition equilibria together with new, simplified theoretical considerations on the effect of strand unravelling on mobility have shown that the gel behavior is predictable for known sequences. A number of the distinctive consequences of the theory and their correspondence with the properties of real molecules have been demonstrated. These include the extremely close cooperative linkage of large blocks of bases into domains, the existence of sharp boundaries between domains, the major role of nearest-neighbor interaction in determining stability, the dependence of domain structures on neighboring and more remote sequences, and the depression of domain melting temperature if the sequence lies at the end of a molecule. New and unusual applications derive from the possibility of separating DNA molecules by properties of their sequence. Exceedingly complex mixtures, such as the sum of all fragments produced by the action of a sixbase specific restriction endonuclease on a complete bacterial genome, can be resolved completely. Additional inserted sequences are easily discerned. The difference of a single base pair in a molecule permits detection and isolation of mutant sequences. The need for full sequential analysis of long molecules for characterization of mutants can be reduced by localizing a change within a small fragment.

Bacteriophage lambda↗

PubMedDate not supplied

Detection of single base substitutions in total genomic DNA.

Certain single base substitutions causing genetic diseases or resulting in polymorphisms linked to mutant alleles, alter a restriction enzyme cleavage site and can therefore be detected in total genomic DNA using DNA blots. Many base substitutions do not lead to an altered restriction site, but these can be detected using synthetic oligonucleotides as hybridization probes if the DNA sequence surrounding the base substitution is known. In the case of beta-thalassaemia, where 22 different single base mutations have been identified, a large number of probes would be required for diagnosis. An approach which was used to detect mutations in viral DNA involves the S1 nuclease treatment of heteroduplexes formed between wild-type and mutant DNA. Although certain single base mismatches are cleaved by S1 nuclease (ref. 11 and T. Shenk, personal communication), many other mismatches examined by this procedure are not cleaved (B. Seed, personal communication; R.M.M., unpublished data). Heteroduplexes between mutant and wild-type subgenomic fragments of double-stranded reovirus RNA migrate slower than the corresponding homoduplexes in polyacrylamide gels containing 7 M urea, but it is not known whether this method is applicable to DNA heteroduplexes containing single base mismatches. Here we describe a procedure that involves the electrophoretic separation of DNA heteroduplexes in a well-characterized gel system. We show that four different human beta-thalassaemia alleles with known single base mutations can be detected with as little as 5 micrograms of total genomic DNA. The method should be useful in the localization and diagnosis of mutations associated with genetic diseases.

Base Sequence↗