The gene coding for ribosomal RNA in Physarum polycephalum exist as a collection of about 200 extrachromosomal DNA molecules 60 kb in size. This rDNA can be purified readily; most of it has been cloned and sequenced. We propose to continue our work on the structure and function of these genes. I. A major fraction of the effort will be devoted to studying two specific DNA binding proteins that we have purified partially. One binds to sequences just upstream of the RNA start site, and the other binds close to the rDNA telomeres. The proteins will be purified further, if possible to homogeneity, and their precise binding sites determined. II. We have discovered a third, strain-specific intron in the 26s coding region of the rDNA. What appears to be a single copy chromosomal sequence homologous to the intron exists in strains not carrying the third intron in their rDNA. The intron will be sequenced, and the chromosomal homologue clond and sequenced as well, to gain clues to their evolutionary relationship. III. The mechanism underlying the single copy, non-Mendelian inheritance of rDNA will be studied, by analysis of rDNA in spores, by a search for destruction and amplification of rDNA in the life cycle, and by testing nucleoli in heterozygous plasmodia for fixation of one or the other rDNA type. IV. The exact locations of the replication origins will be determined by biochemical techniques applied to rDNA enriched for replicating forms. V. The ability of Physarum rDNA to replicate stably in yeast will be studied with particular reference to the questions, Where is replication initiated? and, What sequences are important for the high efficiency of transformation? Preliminary experiments with transient expression assays will be done to test methods for introduction of the rDNA back into Physarum.
