The ultimate goal is to understand how cells balance synthesis and degradative processes to produce a net amount of rRNA. An understanding of ribosome formation and turnover is indispensible for an adequate description of how cells grow and divide. For example, the effects of cell transformation by SV40 virus include both a very early stimulation of cellular rRNA formation and a relative inhibition of the rate of rRNA turnover; both inevitably contribute to the growth advantage of the transformed cell. These studies are designed to analyze rRNA processing and nucleolar structure in detail.
The specific aims i nclude: 1) Definition of processing signals in pre-rRNA, using tests for the transcription and processing of transfected rDNA and mutagenized rDNA in vivo; 2) Measurements of the degree of interdependence of rRNA and r-protein turnover, and of the relative stability of transcribed spacer sequences in vivo and in vitro; 3) characterization of the sequence content and possible functions of non-transcribed spacer rDNA, which we have newly cloned; and 4) fractionation of non-DNA nucleolar components from exhaustively DNase-treated nucleoli (especially pre-ribosomes, nucleolar-specific proteins, and U3 snRNA species). Features of secondary structure of the pre-ribosomes will then be assessed by electron microscopy, and the extent and nature of their association with other nucleolar components examined.
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