PubMed Health⌕ Search

Biomedical subjects

A Hampel

Publications and source records attributed to A Hampel.

10 recordsLinked to original sources
PubMed1992

A method for generating transcripts with defined 5' and 3' termini by autolytic processing.

Plasmids containing both the hammerhead and hairpin ribozyme autocatalytic cassettes were constructed for the purpose of generating RNA transcripts with specific termini at both the 5' and 3' ends. Following transcription, the RNA encoded by these cassettes was capable of intramolecular cleavage. This resulted in the generation of a processed RNA, which was located between the two cassettes, with specifically engineered 5' and 3' ends. The two different ribozymes were selected for their efficient intramolecular cleavage ability and to reduce the possibility of DNA recombination that could occur if identical cassettes were used. An application of this technique was the generation of a processed RNA which was itself a ribozyme, with specific 5' and 3' termini. The ribozyme generated was a hairpin ribozyme specific for a sequence in the gene encoding hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoA reductase). The processed ribozyme was fully catalytically active against an RNA substrate sequence of HMGCoA reductase.

Autolysis↗
PubMed1992

Inhibition of human immunodeficiency virus type 1 expression by a hairpin ribozyme.

Ribozymes are RNAs that possess the dual properties of RNA sequence-specific recognition, analogous to conventional antisense molecules, and RNA substrate destruction via site-specific cleavage. The cleavage reaction is catalytic in that more than one substrate molecule is processed per ribozyme molecule. We have designed a hairpin ribozyme that cleaves human immunodeficiency virus type 1 (HIV-1) RNA in the leader sequence (at nucleotides +111/112 relative to the transcription initiation site). The ribozyme was tested in vitro and gave efficient and specific cleavage of RNA containing the leader sequence. To test the antiviral efficacy of this ribozyme, we have cotransfected into HeLa cells HIV-1 proviral DNA and a plasmid expressing the ribozyme from the human beta-actin promoter. HIV-1 expression was inhibited as measured by p24 antigen levels and reduced Tat activity. The antiviral effect of the ribozyme appears to be specific and results from directed RNA cleavage; activity requires both a target sequence and a functional RNA catalytic center. These results suggest that this HIV-1-directed hairpin ribozyme may be useful as a therapeutic agent.

Base Sequence↗
PubMed1992

Chemical synthesis of RNA using fast oligonucleotide deprotection chemistry.

The exocyclic amine protecting groups in oligonucleotide synthesis which require 8-16 hours at 55 degrees C for deprotection in ammonia have been replaced with more labile base protecting groups (dimethylformamidine for adenine and guanine and isobutyryl for cytosine). Using these fast oligonucleotide deprotecting groups which require 2-3 hours at 55 degrees C for complete deprotection, a new set of cyanoethyl phosphoramidite ribonucleoside monomers and supports has been developed. Ribozymes and substrate RNAs which were synthesized with these phosphoramidites were assayed and were found to have full catalytic (biological) activity.

Base Sequence↗
PubMed1990

'Hairpin' catalytic RNA model: evidence for helices and sequence requirement for substrate RNA.

We have identified the catalytic domain within the sequence of the negative strand of the satellite RNA of tobacco ringspot virus. Minimum energy RNA folding calculations predict a two dimensional model with four major helical regions which are supported by mutagenesis experiments. This model for the catalytic complex consists of a 50 base catalytic RNA and a 14 base substrate RNA folded together in a type of hairpin two dimensional structure. Part of the recognition region between the catalyst and substrate is two helices of 6 bases and 4 bases respectively. Catalytic activity remains when the bases in these two helices are changed but base pairing is maintained. Thus an appropriately engineered 'hairpin' catalyst is capable of cleaving heterologous RNA.

Base Sequence↗
PubMed1989

RNA catalytic properties of the minimum (-)sTRSV sequence.

We have identified an RNA catalytic domain within the sequence of the 359 base long negative-strand satellite RNA of tobacco ringspot virus. The catalytic domain contains two minimal sequences of satellite RNA, a 50-base catalytic RNA sequence, and a 14-base substrate RNA sequence. The catalytic complex of catalytic RNA/substrate RNA represents a structure not previously found in any RNA catalytic reaction described to date. The reaction is truly catalytic since the catalytic RNA has multiple substrate cleavage events and is not consumed during the course of the reaction. A linear relationship is seen between reaction rate and catalytic RNA concentration. The reaction has a Km of 0.03 microM, a kcat of 2.1/min, a temperature optimum of near 37 degrees C, and an energy of activation of 19 kcal/mol.

Base Sequence↗
PubMed1986

Nucleotide sequence and newly formed phosphodiester bond of spontaneously ligated satellite tobacco ringspot virus RNA.

The satellite RNA of tobacco ringspot virus (STobRV RNA) replicates and becomes encapsidated in association with tobacco ringspot virus. Previous results show that the infected tissue produces multimeric STobRV RNAs of both polarities. RNA that is complementary to encapsidated STobRV RNA, designated as having the (-) polarity, cleaves autolytically at a specific ApG bond. Purified autolysis products spontaneously join in a non-enzymic reaction. We report characteristics of this RNA ligation reaction: the terminal groups that react, the type of bond in the newly formed junction and the nucleotide sequence of the joined RNA. The nucleotide sequence of the ligated RNA shows that joining of the reacting RNAs restored an ApG bond. The junction ApG has a 3'-to-5' phosphodiester bond. Thus the net ligation reaction of STobRV (-)RNA is the precise reversal of autolysis. We discuss this new type of RNA ligation reaction and its implications for the formation of multimeric STobRV RNAs during replication.

Base Sequence↗
PubMed1976

Altered leucyl-transfer RNA synthetase from a mammalian cell culture mutant.

Altered leucyl-tRNA synthetase from a mammalian cell culture temperature-sensitive mutant, tsHl, was compared with enzyme from normal wild type Chinese hamster ovary cells. The mutant enzyme had a Km for leucine four times larger than that of wild type and enzyme levels 3-10% that of wild type. The presence of tRNA was necessary during in vitro heating of the mutant enzyme to allow expression of thermolability while the presence of tRNA protected wild type enzyme against thermal inactivation. The tsHl enzyme was stable when heated alone or in the presence of tRNA, leucine, and ATP simultaneously. The mutant's enzymes aminoacylated tRNALeu, tRNAVal, and tRNAIle with fidelity in vitro as determined by cochromatography of the amino-acyl-tRNA isoacceptors on RPC-5 reversed phase chromatography. The mutant failed to show any defect other than the direct formation of leucyl tRNALeu by leucyl-tRNA synthetase.

Adenosine Triphosphate↗