A method for isolation of nuclei containing undegraded RNA from RNAase-rich plasmodia of Physarum polycephalum.
(1) In order to protect the nuclear RNA of Physarum polycephalum plasmodia during cell homogenisation and purification of the nuclei, the following conditions were used: low temperature (-11 degrees C), high pH (8.1-8.9), formaldehyde (2.8%) and spermine (2.3 mM). (2) The efficiency of these RNAase-inhibiting and inactivating conditions is demonstrated by the high molecular weight of the processing products of transcripts from ribosomal genes (11.9, 9.5 and 5.0 kilobases), which were recovered from the isolated nuclei and visualised on agarose gels. (3) Hybridisation experiments with a DNA probe from an actin gene on size-fractionated nuclear RNA (Northern blots) indicate that the transcripts from actin genes are rapidly spliced in P. polycephalum. (4) The nuclear polyadenylated RNA has an average size of about 2.2 kb, which is not significantly larger than the average length of mRNA.
Nothacker
K D
KD
Fachbereich Biologie der Universität Bremen, F.R.G.
Hildebrandt
A
A
D015152:Blotting, Northern; D002458:Cell Fractionation / Q000379:methods; D002467:Cell Nucleus / Q000648:ultrastructure*; D015342:DNA Probes; D005656:Fungal Proteins / Q000235:genetics; D005796:Genes; D005800:Genes, Fungal; D007202:Indicators and Reagents; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D010804:Physarum / Q000235:genetics / Q000648:ultrastructure*; D010957:Plasmids; D012331:RNA, Fungal / Q000235:genetics / Q000302:isolation & purification*
D015342:DNA Probes; D005656:Fungal Proteins; D007202:Indicators and Reagents; D012331:RNA, Fungal