Derivatives of the sunY self-splicing intron efficiently catalyzed the synthesis of complementary strand RNA by template-directed assembly of oligonucleotides. These ribozymes were separated into three short RNA fragments that formed active catalytic complexes. One of the multisubunit sunY derivatives catalyzed the synthesis of a strand of RNA complementary to one of its own subunits. These results suggest that prebiotically synthesized oligonucleotides might have been able to assemble into a complex capable of self-replication.

Understanding the mechanisms by which ribozymes catalyse chemical reactions requires a detailed knowledge of their structure. The secondary structure of the group I introns has been confirmed by comparison of over 70 published sequences, by chemical protection studies, and by genetic experiments involving compensatory mutations. Phylogenetic data can also be used to identify tertiary interactions in RNA molecules. This was first done by Levitt, who predicted tertiary interactions in transfer RNA, which were subsequently confirmed by X-ray crystallography. More recently, sequence comparison data have been used to predict tertiary interactions in ribosomal RNA. We have searched a complete alignment of the core regions of group I introns for evolutionary covariations that could not be ascribed to classical Watson-Crick or wobble base pairings. Here we describe two examples of phylogenetic covariation that are most simply explained by postulating hydrogen-bonded base-triples similar to those found in tRNA. Genetic experiments with the Tetrahymena and sunY introns confirm the importance of these interactions for the structure of the ribozyme.

No abstract

Recombination walkdng is based on the genetic selection of specific human clones from a yeast artificial chromosome (YAC) library by homologous recombination. The deshied clone is selected from a pooled (unordered) YAC library, eliminating labor-intensive steps typically used in organizing and maintaining ordered YAC libraries. Recombination walking represents an efficient approach to library screening and is well suited for chromosome-walking approaches to the isolation of genes associated with common dieases.