Isolation of polysomes from Nostoc sp. MAC and translation of messenger RNA in a heterologous cell-free system.
A method has been established which isolated polysomes from the lysozyme/EDTA-shocked cyanobacterium, Nostoc sp. MAC. In a typical preparation the total recovery of RNA as polysomes was 83%, in which 77% of the polysome fraction was present at sizes greater than 5-mers and 23% as 2-4-mers. Messenger RNA isolated from such a preparation of polysomes produced a 10-fold stimulation in the incorporation of [35S]methionine into polypeptides by a cell-free system of Escherichia coli. The in vitro-synthesized polypeptides were analysed on an SDS-polyacrylamide gradient gel together with in vivo-labelled proteins of Nostoc sp. MAC: seven polypeptides co-migrated with the in vivo-synthesized products. This is the first report of the expression of cyanobacterial messenger RNA in a heterologous cell-free system from E. coli; the efficiency of the system is discussed.
D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient; D000458:Cyanobacteria / Q000032:analysis* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D011132:Polyribosomes / Q000032:analysis*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*
D012333:RNA, Messenger