In situ hybridisation in filamentous fungi using peptide nucleic acid probes.
Fluorescent DNA and peptide nucleic acid (PNA) probes were used for in situ hybridisations in colonies of Schizophyllum commune and Aspergillus niger. DNA probes for 18S rRNA did not diffuse through the cell wall after mild chemical fixation. After permeabilising the cell wall with lysing enzymes or slow freezing and embedding, hybridisation was still poor and not reproducible. In contrast, PNA probes did diffuse through the cell wall after mild chemical fixation and reproducible fluorescent signals were obtained. The rRNA signal was most intense in the apical compartment of hyphae of S. commune. Within this compartment, the signal was lower at the extreme apex. Apparently, ribosomes are unevenly distributed in hyphae. In S. commune, the mRNA of the SC3 gene was also detected with a PNA probe. The ratio between 18S rRNA and SC3 mRNA signals were variable between hyphae and their compartments. This is the first report of using PNA probes for in situ hybridisation of mRNA in fungi. The method provides a powerful tool to study gene expression.
Teertstra
Wieke R
WR
Microbiology, Institute of Biomembranes, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Lugones
Luis G
LG
Wösten
Han A B
HA
D001234:Aspergillus niger / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods*; D005800:Genes, Fungal / Q000235:genetics; D025301:Hyphae / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D020135:Peptide Nucleic Acids / Q000378:metabolism*; D012331:RNA, Fungal / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D012337:RNA, Ribosomal, 18S / Q000032:analysis; D012567:Schizophyllum / Q000235:genetics*
D020135:Peptide Nucleic Acids; D012331:RNA, Fungal; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S