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Biomedical subjects

G F Joyce

Publications and source records attributed to G F Joyce.

15 recordsLinked to original sources
PubMed1992

Directed evolution of an RNA enzyme.

An in vitro evolution procedure was used to obtain RNA enzymes with a particular catalytic function. A population of 10(13) variants of the Tetrahymena ribozyme, a group I ribozyme that catalyzes sequence-specific cleavage of RNA via a phosphoester transfer mechanism, was generated. This enzyme has a limited ability to cleave DNA under conditions of high temperature or high MgCl2 concentration, or both. A selection constraint was imposed on the population of ribozyme variants such that only those individuals that carried out DNA cleavage under physiologic conditions were amplified to produce "progeny" ribozymes. Mutations were introduced during amplification to maintain heterogeneity in the population. This process was repeated for ten successive generations, resulting in enhanced (100 times) DNA cleavage activity.

Animals↗
PubMed1992

Randomization of genes by PCR mutagenesis.

A modified polymerase chain reaction (PCR) was developed to introduce random point mutations into cloned genes. The modifications were made to decrease the fidelity of Taq polymerase during DNA synthesis without significantly decreasing the level of amplification achieved in the PCR. The resulting PCR products can be cloned to produce random mutant libraries or transcribed directly if a T7 promoter is incorporated within the appropriate PCR primer. We used this method to mutagenize the gene that encodes the Tetrahymena ribozyme with a mutation rate of 0.66% +/- 0.13% (95% C.I.) per position per PCR, as determined by sequence analysis. There are no strong preferneces with respect to the type of base substituion. The number of mutations per DNA sequence follows a Poisson distribution and the mutations are randomly distributed throughout the amplified sequence.

Animals↗
PubMed1991

The rise and fall of the RNA world.

It is generally believed that there was a time when life on earth was based on RNA rather than on DNA and protein. Considering the relevant evidence from geophysics, geology, paleobiology, and molecular biology, it is possible to set the time frame for the existence of RNA-based life to a 400 million year interval beginning 4.0 to 4.2 billion years ago and ending 3.6 to 3.8 billion years ago. The minimum level of biochemical complexity that existed during this time consists of those functions necessary for the establishment and maintenance of an RNA-based evolving system, namely, an RNA unwinding activity, an RNA replicase activity, and a primitive biosynthetic apparatus leading to enrichment of the local environment with activated D mononucleotides.

Biological Evolution↗
PubMed1991

RNA today. Molecular Evolution of Introns and Other RNA Elements: a Keystone Symposium, Taos, NM, USA, February 2-8, 1991.

RNA research is alive and well. The joyride for those studying the biochemistry and molecular biology of RNA continues, although perhaps not at the thrill-a-month pace of recent years. The Keystone Symposium provided an opportunity to gain deeper insight into RNA-based biological phenomena by attempting to place current research in an evolutionary context. In this sense the meeting was an unqualified success. The meeting participants, having been warmed by the New Mexico sun and the chile-laden cuisine, now return to their laboratories determined to pursue not only the details of RNA biochemistry and molecular biology, but also the evolutionary implications of their work.

Introns↗
PubMed1990

Minimum secondary structure requirements for catalytic activity of a self-splicing group I intron.

We have completed a comprehensive deletion analysis of the Tetrahymena ribozyme in order to define the minimum secondary structure requirements for phosphoester transfer activity of a self-splicing group I intron. A total of 299 nucleotides were removed in a piecewise fashion, leaving a catalytic core of 114 nucleotides that form 7 base-paired structural elements. Among the various deletion mutants are a 300-nucleotide single-deletion mutant and a 281-nucleotide double-deletion mutant whose activity exceeds that of the wild type when tested under physiologic conditions. Consideration of those structural elements that are essential for catalytic activity leads to a simplified secondary structure model of the catalytic core of a group I intron.

Animals↗
PubMed1990

Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA.

The discovery of RNA enzymes has, for the first time, provided a single molecule that has both genetic and catalytic properties. We have devised techniques for the mutation, selection and amplification of catalytic RNA, all of which can be performed rapidly in vitro. Here we describe how these techniques can be integrated and performed repeatedly within a single reaction vessel. This allows evolution experiments to be carried out in response to artificially imposed selection constraints. We worked with the Tetrahymena ribozyme, a self-splicing group I intron derived from the large ribosomal RNA precursor of Tetrahymena thermophila that catalyses sequence-specific phosphoester transfer reactions involving RNA substrates. It consists of 413 nucleotides, and assumes a well-defined secondary and tertiary structure responsible for its catalytic activity. We selected for variant forms of the enzyme that could best react with a DNA substrate. This led to the recovery of a mutant form of the enzyme that cleaves DNA more efficiently than the wild-type enzyme. The selected molecule represents the discovery of the first RNA enzyme known to cleave single-stranded DNA specifically.

Animals↗
PubMed1989

Amplification, mutation and selection of catalytic RNA.

RNA, by virtue of its genotypic and phenotypic properties, is a suitable substrate for molecular evolution in the laboratory. We have developed techniques for the rapid amplification, mutation and selection of catalytic RNA. By combining these techniques in an iterative fashion, we are attempting to construct an RNA-based evolving system. Such a system could be used to explore the catalytic potential of RNA.

Biological Evolution↗
PubMed1989

Catalytic activity is retained in the Tetrahymena group I intron despite removal of the large extension of element P5.

We have made sizeable internal deletions within the self-splicing group I intron of Tetrahymena thermophila. Deletions were made in a piecewise manner in order to remove secondary structural elements thought to be extraneous to the catalytic center of the molecule. The resulting deletion mutants retain self-splicing activity, albeit under modified reaction conditions that enhance duplex stability. Considering those portions of the molecule that can be deleted without a loss of catalytic activity, one is left with a catalytic center of approximately 130 nucleotides that is solely responsible for the molecule's activity.

Animals↗
PubMed1989

RNA evolution and the origins of life.

The evolution of RNA is likely to have played an important role in the very early history of life on Earth but it is doubtful that life began with RNA. Consideration of what came before RNA must take into account relevant information from geochemistry, prebiotic chemistry and nucleic acid biochemistry.

Biological Evolution↗
PubMed1989

A novel technique for the rapid preparation of mutant RNAs.

We have developed a novel in vitro mutagenesis technique that allows us to introduce mutations at the level of double-stranded DNA and then transcribe the mutant DNA directly. The technique is useful for those wishing to produce recombinant RNA, particularly if the desired recombinant is the result of an insertion or deletion. It is also useful for the preparation of 3'-truncated RNAs with a defined end. The technique is not dependent on the presence of a convenient restriction site within the target gene, and does not involve construction of a clone or amplification of the mutant DNA within a bacterial host. It is intended as a simple and rapid method for the preparation of roughly 100-200 pmol of mutant RNA, which would be sufficient for obtaining sequence information and assessing the functional consequences of the mutation.

Animals↗
PubMed1988

Non-enzymatic template-directed synthesis on RNA random copolymers. Poly(C,A) templates.

Poly(C,A) random copolymer templates direct the oligomerization of 2-MeImpG (2-MeImpX is the 5'-phospho-2-methylimidazolide of the nucleoside X) and 2-MeImpU, resulting in the production of a variety of oligo (G,U)s. This reaction is less efficient than comparable reactions involving poly(C,U) or poly(C,G) templates. The efficiency of monomer incorporation into newly synthesized oligomers is lower for 2-MeImpU than 2-MeImpG, and cannot be improved by increasing the concentration of 2-MeImpU relative to 2-MeImpG. This suggests that RNA templates containing runs of consecutive adenine residues would not be suitable for use in a chemical self-replicating system. The distribution of oligomeric products can be characterized in detail using high-pressure liquid chromatography on an RPC-5 column. Oligomers are separated on the basis of chain length, base composition, and phosphodiester-linkage isomerism. Oligomers up to about the 13-mer, with base composition Gn, Gn-1, U, and Gn-2, U2, have been identified.

Chromatography, High Pressure Liquid↗
PubMed1987

The case for an ancestral genetic system involving simple analogues of the nucleotides.

The idea that the first living systems on earth were based on self-replicating RNA molecules has recently become popular as a result of the discovery of ribozymes. However, there are several major problems associated with the prebiotic synthesis of ribonucleotides. In addition, there is the newly recognized problem of enantiomeric cross-inhibition, whereby template-directed polymerization involving one enantiomer of RNA is inhibited strongly by the presence of the other enantiomer. Here we propose that RNA was preceded in the evolution of life by a polymer constructed from flexible, acyclic, probably prochiral nucleotide analogues that were synthesized readily on the primitive earth. Several potentially prebiotic nucleotide analogues are considered in this context, and some of the consequences of this proposal are discussed.

Models, Genetic↗
PubMed1986

Non-enzymic template-directed synthesis on RNA random copolymers. Poly(C, G) templates.

Poly(C, G) random copolymer templates direct the oligomerization of 2-Me-ImpG and 2-MeImpC, resulting in the production of a variety of oligo(G, C)s. The efficiency of monomer incorporation into newly synthesized oligomers is greater for 2-MeImpG than for 2-MeImpC, and decreases for both monomers as the guanine content of the template increases. The relatively low efficiency of oligomerization on guanine-rich templates is largely a consequence of intra- and intermolecular template self-structure. The problem of template self-structure is clearly a major obstacle to the development of a system of self-replicating polynucleotides. The distribution of oligomeric products can be characterized in detail using high-pressure liquid chromatography on an RPC-5 column. Oligomers are separated on the basis of chain length, base composition and phosphodiester-linkage isomerism. Oligomers up to about the 12-mer, with base composition Gn, Gn-1C and Gn-2C2, have been identified. The 3' to 5' regiospecificity of the products is high, particularly for oligomers with base composition Gn.

Chromatography, High Pressure Liquid↗