An improved method for extraction of mRNA and protein from the fungus Chaetomium gracile with anhydrous hydrogen fluoride.
We previously reported that DNAs directly applicable to restriction analyses and transformation of Escherichia coli can be extracted from fungi 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) FEBS Lett. 220, 383-386]. In the present investigation, 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 anhydrous hydrogen fluoride (HF) treatment for a short time (1 min) at 0 degrees C. The extracted poly(A)-enriched mRNA and proteins were fully intact: the mRNA purified by messenger-activated paper with poly(U) directed not only the incorporation of [3H]glycine into polypeptides but also the synthesis in a rabbit reticulocyte lysate cell-free system of proteins reactive to antibodies against the soluble fraction extracted from the fungus by HF. Analyses by gel filtration and polyacrylamide electrophoresis under native conditions showed that dextranase extracted from the fungus by HF under the same conditions had the same molecular weight and electrophretic mobility as the enzyme excreted into the medium. This suggests that the mRNAs and proteins extracted by this method are also applicable to protein synthesis directed in a cell-free system and to enzyme purification from a fungus insusceptible to lytic enzymes. This method provides a pure preparation of mRNA within 5 h and starting materials for protein purification within 1 h.
Oshiro
S
S
Department of Biochemical Genetics, Tokyo Medical and Dental University.
Katsura
N
N
D000818:Animals; D002473:Cell Wall / Q000187:drug effects; D002474:Cell-Free System; D002597:Chaetomium / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics; D005656:Fungal Proteins / Q000302:isolation & purification*; D006858:Hydrofluoric Acid; D012331:RNA, Fungal / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000302:isolation & purification*; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000187:drug effects
D005656:Fungal Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D006858:Hydrofluoric Acid