Stoichiometric analysis of protein- and nucleic acid-based structures in the cell nucleus.
We describe a method to image selectively the protein-based architecture in the eukaryotic cell nucleus using nitrogen and phosphorus mapping. In addition, we describe a method to determine total mass as well as stoichiometric relationships between protein and RNA. This method is illustrated using particulate structures in the nucleus called interchromatin granules. In so doing, we demonstrate that these granules contain heterogeneous nuclear RNA, and have an average protein and RNA content of 3.094 and 1.672 MDa, respectively. We also tested the sensitivity of phosphorus detection by exogenously applying purified duplex DNA to the surfaces of thin sections, and have shown that structures as small as single molecules of duplex DNA can be detected in situ using these electron spectroscopic imaging techniques.
Department of Cell Biology and Anatomy, University of Calgary, Alberta, Canada. bazett@acs.ucalgary.ca
D P
DP
Bazett-Jones
M J
MJ
Hendzel
M J
MJ
Kruhlak
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry* / Q000648:ultrastructure; D002843:Chromatin / Q000378:metabolism / Q000648:ultrastructure; D005347:Fibroblasts; D008854:Microscopy, Electron / Q000379:methods; D018668:Muntjacs; D009584:Nitrogen / Q000378:metabolism; D009687:Nuclear Proteins / Q000032:analysis*; D010758:Phosphorus / Q000378:metabolism; D015138:RNA, Nuclear / Q000032:analysis*; D012270:Ribosomes / Q000378:metabolism
D002843:Chromatin; D009687:Nuclear Proteins; D015138:RNA, Nuclear; D010758:Phosphorus; D009584:Nitrogen