This application centers around two questions: (1) How do eucaryotic cells regulate the amount of synthesis of particular proteins in response to changing metabolic needs? and (2) How are genes which are present in many copies in each nucleus kept identical in the face of changes by mutation? The ultimate aim of this work is a better understanding of how genes manage the metabolic housekeeping of the cell, and how they save themselves from mutational deterioration. We have studied how Neurospora crassa, a filamentous fungus, recognizes that it has slipped into a state of phosphorus deficiency and starts making enzymes that help it wring the remaining phosphorus out of the growth medium. The organism also cuts back the synthesis of ribosomal RNA, an essential but deferrable process that is very costly in terms of phosphorus. We want to know genes regulate these changes. We have mutants that mis-regulate this adaptive process, either making the enzymes when they shouldn't, or failing to make them when they should. I propose isolating the structural and regulatory genes of phosphorus metabolism by recombinant DNA technology. Having the genes on hand as ordinary chemical reagents should allow us to look at how regulation of enzyme synthesis works in the normal organism. The question of how multiple copy genes resist mutational deterioration is also being approached with recombinant DNA technology. We have cloned about 25 genes for 5S RNA. These genes specify several appreciably different RNA sequences (isotypes). Some unidentified correction mechanism prevents the sequences from diverging beyond these few permitted isotypes. I propose experiments that ask if the different isotypes are each useful in some special way, and whether synthesis of each isotype is separately regulated. I also suggest ways to detect """"""""correction"""""""" of aberrant 5S DNA as an evolutionary alternative to death of the mutant cell.
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