Comparative pyrimidine- and purine-specific RNAse-gold labeling on pancreatic acinar cells and isolated hepatocytes.
We applied the enzyme-gold approach to investigate the potential of various ribonucleases displaying different affinities for ultrastructural localization of particular RNA molecules. Five specific ribonucleases were used: three from a pancreatic source, RNAses A, B, and S with affinities for pyrimidine bases; and two from Aspergillus oryzae, RNAses T1 and T2 specific for purine bases. Conditions required for preparing each RNAse-gold complex, as well as for obtaining specific labelings, were determined. Application of the probes on thin sections of pancreatic acinar cells yielded labeling patterns that differed according to the enzyme used. Pancreatic RNAses labeled mostly the rough endoplasmic reticulum and the nucleolus, whereas fungal RNAses labeled more intensely the interchromatin space and the nucleolus, the rough endoplasmic reticulum being labeled to a lesser extent. Areas rich in interchromatin granules were intensely labeled by the RNAses T1 and T2. This was confirmed on DRB-treated hepatocytes, which displayed large clusters of interchromatin granules. Perichromatin granules were labeled by the RNAse A- and T1-gold complexes. These results provide a strong indication for the presence of RNA molecules in both types of granules. Nuclear pores were labeled, particularly by the RNAses T1 and T2, thus supporting the hypothesis for the site of RNA transit between nucleus and cytoplasm. The differences in patterns of labeling among the various enzyme-gold complexes could be related to differences in affinities. The use of a panel of specific RNAses, displaying different affinities, could thus allow for the topographical distribution of particular RNA molecules according to their relative content of specific bases.
Cheniclet
C
C
Département d'Anatomie, Université de Montréal, Québec, Canada.
Bendayan
M
M
D000818:Animals; D002478:Cells, Cultured; D006046:Gold / Q000378:metabolism; D006651:Histocytochemistry / Q000379:methods; D008099:Liver / Q000166:cytology* / Q000201:enzymology / Q000648:ultrastructure; D008854:Microscopy, Electron; D010179:Pancreas / Q000166:cytology* / Q000201:enzymology / Q000648:ultrastructure; D011687:Purines / Q000378:metabolism*; D011743:Pyrimidines / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D015138:RNA, Nuclear / Q000378:metabolism; D051381:Rats; D012260:Ribonucleases / Q000378:metabolism*
D011687:Purines; D011743:Pyrimidines; D015138:RNA, Nuclear; D012313:RNA; D006046:Gold; D012260:Ribonucleases