Highly repetitive, simple sequence satellite DNAs are common in eukaryotic genomes, despite the fact that they are typically inert transcriptionally. The 330 base pair, tandemly repeated satellite 2 sequence in the newt, Notophthalmus viridescens, differs in that it is transcribed into a series of stable, cytoplasmic transcripts. In addition, these transcripts have the ability to undergo site- specific, self-catalyzed hydrolysis in vitro. Cleavage occurs within a sequence that has significant homology to the self- cleavage sites of a number of infectious RNAs found in plants. The mechanism of self-cleavage, the function of the satellite 2 transcripts, and the relation of these transcripts to infectious RNAs will be investigated with the ultimate goal of understanding the evolution and function of at least one class of eukaryotic satellite DNA. The mechanism of in vitro self-catalysis will be analyzed by a complete biochemical characterization of the cleavage products. Regions involved in secondary- and tertiary-structural interactions will then be determined and subsequently disrupted by in vitro mutagenesis to determine their importance to the cleavage reaction. To investigate the role of self-catalysis in the formation of the in vivo transcripts, the primary structure of each of the different satellite 2 transcripts will be determine. Factors which may be required for cleavage in vivo will be sought in oocytes by performing the cleavage reactions in the presence of ovary extracts and by injecting synthetic transcript into oocytes. To study the formation of transcripts in somatic cells, a cultured cell transformation system will be developed for the introduction of modified satellite 2 sequences into oviduct cell lines. The possible role of infectious RNAs in the evolution of the eukaryotic genome will be explored by probing DNA from a variety of organisms for the conserved cleavage site. Any DNA possessing this sequence will be cloned, and transcripts from this DNA will be tested for the ability to self-cleave.
