AN IMPROVED METHOD FOR EXTRACTION OF MESSENGER-RNA AND PROTEIN FROM THE FUNGUS CHAETOMIUM-GRACILE WITH ANHYDROUS HYDROGEN-FLUORIDE

We previously reported that DNAs directly applicable to restriction an alyses and transformation of Escherichia coli can be extracted from fu ngi and yeasts by use of anhydrous hydrogen fluoride (HF) under a mild condition, 5 min at 0 degrees C [Oshiro, S., Katsura, N., Kitada, K., and Gunge, N. (1987) FEES Lett. 220, 383-386]. In the present investi gation, we examined whether this improved method is also applicable to extraction of RNA and protein from the fungus Chaetomium gracile. The RNA and protein were effectively extracted from the fungus after anhy drous hydrogen fluoride (HF) treatment for a short time (1 min) at 0 d egrees C. The extracted poly(A)-enriched mRNA and proteins were fully intact: the mRNA purified by messenger-activated paper with poly(U) di rected not only the incorporation of [H-3]glycine into polypeptides bu t also the synthesis in a rabbit reticulocyte lysate cell-free system of proteins reactive to antibodies against the soluble fraction extrac ted from the fungus by HF. Analyses by gel filtration and polyacrylami de electrophoresis under native conditions showed that dextranase extr acted from the fungus by HF under the same conditions had the same mol ecular weight and electrophretic mobility as the enzyme excreted into the medium. This suggests that the mRNAs and proteins extracted by thi s method are also applicable to protein synthesis directed in a cell-f ree system and to enzyme purification from a fungus insusceptible to l ytic enzymes. This method provides a pure preparation of mRNA within 5 h and starting materials for protein purification wthin 1 h.