8 SUBCELLULAR PARTICLES 



tion for speculation; so much so that Sjostrand, in a recent review, pleads that 

 electron microscopists guard against making the field a 'science of guesses' (48). 

 However one may feel about such speculation, it is apparent that at best elec- 

 tron microscopy alone remains purely descriptive and essentially morphological. 

 It needs to be supplemented by techniques providing biochemical information. 



RADIOAUTOGRAPHY 



The most direct approach to m vivo biochemical events within particulates is 

 that of radioautography. Dr. Taylor, in this volume, shows the impressive cyto- 

 logical resolution now possible with this technique (54). I will refer to only two 

 reports in the literature. 



The first is that of the now classic experiments of Goldstein and Plant (15). 

 These combine radioautography with the microdissection of living cells possible 

 with Ameba and other large cells. 



By feeding Ameba microorganisms reared on P^"-orthophosphate, its nucleus 

 is made to incorporate P^- into its ribonucleic acid (RNA). The fate of this radio- 

 active nuclear RNA can then be followed when transplanted into an enucleated 

 half of a nonradioactive Ameba. After some 62 hours, considerable radioactivity 

 has moved into the cytoplasm. That this is not a random loss from the nucleus 

 is shown by transplanting a radioactive ('hot') nucleus into a whole nonradio- 

 active ('cold') Ameba. While much radioactivity moves into the cytoplasm of 

 the 'cold' Ameba, none goes into the 'cold' nucleus. 



From indirect evidence, such as effects of ribonuclease digestion, the conclu- 

 sion is drawn that the material moving from nucleus to cytoplasm is RNA or a 

 material much like it. 



Radioautography has another unportant aspect. It lends itself to fairly good 

 quantitation, as may be illustrated by the work of McMaster-Kaye and Taylor 

 (24). From the time curves of the number of reduced grains appearing over 

 cytological structures, the authors concluded that P^~ is incorporated into nu- 

 cleolar RNA considerably earlier than into cytoplasmic RNA. 



Even radioautography, however, is not free of difficulties. Aside from de- 

 pendence upon indirect methods for the identification of the substance in which 

 the radioactivity is incorporated, there are the difficulties that soluble radioactive 

 substances are lost in the methods of tissue preparation currently employed, that 

 ignorance of the nature and magnitude of precursor pools may make interpreta- 

 tion difficult, and that incorporation sites need not necessarily be the sites of 

 function. 



ISOLATION OF SUBCELLULAR PARTICLES BY CENTRIFUGATION 



Despite all its uncertainties and limitations, the isolation of subcellular frac- 

 tions by centrifugation of homogenates has provided the main foundation of 



