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Joyce, G. F.

Publications and source records attributed to Joyce, G. F..

28 records · Page 2
All sourcesNASA NTRSDOE OSTI
NASA NTRS1993

Evolution in vitro of an RNA enzyme with altered metal dependence

The Tetrahymena group I ribozyme catalyses a sequence-specific phosphodiester cleavage reaction on an external RNA oligonucleotide substrate in the presence of a divalent metal cation cofactor. This reaction proceeds readily with either Mg2+ or Mn2+, but no detectable reaction has been reported when other divalent cations are used as the sole cofactor. Cations such as Ca2+, Sr2+ and Ba2+ can stabilize the correct folded conformation of the ribozyme, thereby partially alleviating the Mg2+ or Mn2+ requirement. But catalysis by the ribozyme involves coordination of either Mg2+ or Mn2+ at the active site, resulting in an overall requirement for one of these two cations. Here we use an in vitro evolution process to obtain variants of the Tetrahymena ribozyme that are capable of cleaving an RNA substrate in reaction mixtures containing Ca2+ as the divalent cation. These findings extend the range of different chemical environments available to RNA enzymes and illustrate the power of in vitro evolution in generating macromolecular catalysts with desired properties.

NASA Discipline Exobiology↗
NASA NTRS1993

Evolution in vitro: analysis of a lineage of ribozymes

Background: Catalytic RNAs, or ribozymes, possessing both a genotype and a phenotype, are ideal molecules for evolution experiments in vitro. A large, heterogeneous pool of RNAs can be subjected to multiple rounds of selection, amplification and mutation, leading to the development of variants that have some desired phenotype. Such experiments allow the investigator to correlate specific genetic changes with quantifiable alterations of the catalytic properties of the RNA. In addition, patterns of evolutionary change can be discerned through a detailed examination of the genotypic composition of the evolving RNA population. Results: Beginning with a pool of 10(13) variants of the Tetrahymena ribozyme, we carried out in vitro evolution experiments that led to the generation of ribozymes with the ability to cleave an RNA substrate in the presence of Ca2+ ions, an activity that does not exist for the wild-type molecule. Over the course of 12 generations, a seven-error variant emerged that has substantial Ca(2+)-dependent RNA-cleavage activity. Advantageous mutations increased in frequency in the population according to three distinct dynamics--logarithmic, linear and transient. Through a comparative analysis of 31 individual variants, we infer how certain mutations influence the catalytic properties of the ribozyme. Conclusions: In vitro evolution experiments make it possible to elucidate important aspects of both evolutionary biology and structural biochemistry on a reasonable short time scale.

Non-NASA Center↗
NASA NTRS1993

Climbing Darwin's ladder

The work of Bartel and Szostak, in which RNA molecules were selected to enhance the ability to catalyze a reaction similar to a step in protein-catalyzed RNA replication, is discussed. An important aspect of this experiment was the ability to reach a high level of functional organization in ten evolutionary steps. Further steps necessary to obtain an RNA enzyme with RNA replicase activity include performing the reaction with mononucleoside 5'-triphosphates, generalizing the reaction to include a variety of sequences without loss of template-dependent specificity, and overcoming template self-structure that could prevent some regions from being copied efficiently.

NASA Discipline Exobiology↗
NASA NTRS1991

Controlled evolution of an RNA enzyme

It is generally thought that prior to the origin of protein synthesis, life on earth was based on self-replicating RNA molecules. This idea has become especially popular recently due to the discovery of catalytic RNA (ribozymes). RNA has both genotypic and phenotypic properties, suggesting that it is capable of undergoing Darwinian evolution. RNA evolution is likely to have played a critical role in the early history of life on earth, and thus is important in considering the possibility of life elsewhere in the solar system. We have constructed an RNA-based evolving system in the laboratory, combining amplification and mutation of an RNA genotype with selection of a corresponding RNA phenotype. This system serves as a functional model of a primitive organism. It can also be used as a tool to explore the catalytic potential of RNA. By altering the selection constraints, we are attempting to modify the substrate specificity of an existing ribozyme in order to develop ribozymes with novel catalytic function. In this way, we hope to gain a better understanding of RNA's catalytic versatility and to assess its suitability for the role of primordial catalyst. All of the RNA enzymes that are known to exist in contemporary biology carry out cleavage/ligation reactions involving RNA substrates. The Tetrahymena ribozyme, for example, catalyzes phosphoester transfer between a guanosine containing and an oligopyrimidine containing substrate. We tested the ability of mutant forms of the Tetrahymena ribozyme to carry out a comparable reaction using DNA, rather than RNA substrate. An ensemble of structural variants of the ribozyme was prepared and tested for their ability to specifically cleave d(GGCCCTCT-A3TA3TA) at the phosphodiester bond following the sequence CCCTCT. We recovered a mutant form of the enzyme that cleaves DNA more efficiently than does the wild-type. Beginning with this selected mutant we have now scattered random mutations throughout the ribozyme and have begun an evolutionary search to further expand the catalytic repertoire of RNA.

Joyce, G. F.↗
NASA NTRS1984

Chiral selection in poly(C)-directed synthesis of oligo(G)

An experimental study is reported which shows that poly(C)-directed oligomerization of activated guanosine mononucleotides proceeds readily if the monomers are of the same optical handedness as the template, and is far less efficient if the monomers are of the opposite handedness. However, in template-directed reactions with a racemic mixture, monomers of the opposite handedness to the template are incorporated as chain terminators at the 2'(3') end of the products. This inhibition raises an important problem for many theories of the origin of life.

Joyce, G. F.↗
NASA NTRS1984

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

Random copolymer templates containing cytosine and uracil in ratios of 3:1 and 1:1 are used to explore the optimum conditions for efficient synthesis of guanine and adenine-containing oligonucleotides. The experimental procedure is described, including the preparation of mononucleoside 5'-phospho-2-methylimidazolides and random copolymers, the template-directed oligomerization, the removal and reintroduction of mononucleotides in interrupted reactions, the determination of oligomerization efficiency, the alkaline and enzymatic hydrolysis of reaction products, and the column chromatography. Results are presented and discussed for the dependence of adenine incorporation on the formation of short oligo(G)s, optimization of incorporation efficiencies by adjusting monomer concentrations, the characterization of oligomeric product distribution, and the regiospecificity of adenine incorporation. The prebiotic significance of the results is assessed.

Joyce, G. F.↗
NASA NTRS1984

Template-directed synthesis on the pentanucleotide CpCpGpCpC

Experiments in which CpCpGpCpC is used as a template to facilitate the co-oligomerization of 2-MeImpG and 2-MeImpC are described. It is shown that 3' to 5' prime-linked pGpGpCpGpG, whose sequence is complementary to that of the template, is substantially the most adundant pentameric product of the template-directed reaction. The yield of pGpGpCpGpG is never large (less than 20 percent), presumably becauase off-template reactions consume template-directed products. Thus pGpGpCpGpG is converted to the various isomers of G5C and G4C2 by off-template terminal addition of G or C. The 3' to 5' isomer of GpG is elongated on the template to give GpGpC, GpGpCpG, and GpGpCpGpG, while the 2' to 5' isomer does not initiate the synthesis of detectable amounts of longer oligomers.

Inoue, T.↗