Polyamine-mediated phosphorylation of a nucleolar protein from Physarum polycephalum that stimulates rRNA synthesis

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Abstract
An acidic nucleolar phosphoprotein with a subunit MW of 70,000 was purified as an apparent dimer of 139,000 from isolated nuclei of the slime mold P. polycephalum. The protein was purified without the aid of strong dissociating agents after its selective phosphorylation in isolated nuclei by polyamine-mediated reaction. Its amino acid composition resembled that of a nucleolar phosphoprotein from Novikoff [mouse] hepatoma ascites cells. The phosphoprotein stimulated rRNA synthesis 5-fold by RNA polymerase I within a nucleolar, ribosomal deoxyribonucleoprotein complex isolated from nucleoli of P. polycephalum. It was also identified as a component of the complex. It bound with high affinity and specificity to the palindromic ribosomal DNA of 38 .times. 106 MW from P. polycephalum, which contained 2 coding sequences for 5.8S, 19S and 26S rRNA. It also bound to 3 fragments of ribosomal DNA of MW 21.2 .times. 106, 17.1 .times. 106 and 8.1 .times. 106, prepared by cleavage with restriction endonucleases HindIII, PstI and BamHI, respectively. All of these fragments included the symmetry axis of the palindromic ribosomal DNA. The phosphoprotein that had been treated with alkaline phosphatase-agarose to hydrolyze the phosphate groups did not stimulate transcription and did not bind to ribosomal DNA or to the restriction fragments indicated. Thus, a specific phosphoprotein with the capacity to stimulate transcription of a specific set of genes in a eukaryote was isolated. This phosphoprotein may specifically regulate functions of ribosomal DNA in a manner dependent on its degree of phosphorylation.