Improved technique for the recovery of poly a( )polysomes from physarum polycephalum macroplasmodia, 1986

Physarum polycephalum plasmodia contain highly active endogenous ribonucleases during this phase of their life cycle. Negating the degradative activities of these enzymes has been a serious impediment to the recovery of intact gene and gene products from this developmentally unique organism. This report demonstrates definitive procedures for the recovery of poly A ( ) polysomes from synchronous S phase plasmodia surface cultures using a combination of three potent ribonuclease inhibitors. Total cytoplasmic polysomes were selectively purified on oligo (dT) cellulose affinity columns and poly A ( ) RNA was resolved into multiple discrete fractions using sodium dodecyl sulphate polyacrylamide gel electrophoresis. Polysome integrity, for both total and poly A ( ) RNA, was assessed by measuring messenger capacity to direct the incorporation of radiolabelled amino acids into protein. Experimental results from this study support the contention that exogenous ribonuclease contamination can be effectively minimized through the pre treatment of all glassware and solutions with Diethyl Pyrocarbonate (DEPC). In addition, polysomes extracted in the presence of high ionic strength buffer containing the divalent chelator, Ethylene Glyco bis ( g Amino Ethyl Ether)N,N' Tetraacetic Acid (EGTA) and the competitive ribonuclease inhibitor, Vanadyl Ribonucleoside Complex (VRC), showed both improvements in RNA yield and iji vitro translation activity. These findings strongly suggest that improved methods for the isolation and recovery of poly A ( ) polysomes from Physarum polycephalum macroplasmodia have been developed