148 ALEXANDER L. DOUNCE 



5. HiSTOCHEMICAL STUDIES OF NUCLEOLI 



Almost all histochemical studies of nucleoli have led to the belief that 

 these intranuclear inclusion bodies contain a considerable amount of PNA. 

 A ring of Feulgen-positive material is often found around the nucleolus, 

 however, and workers have reported Feulgen-positive nucleoli. 



As an example of histochemical studies of nucleoli, some work of Pollister 

 and Leuchtenberger may be cited. '^■' These investigators have studied the 

 nucleoli of the maize (Zea mays), which they term plasmosomes. (This term 

 is sometimes used to designate nucleoli, apparently because the latter have 

 staining properties more similar to those of cytoplasm than to those of other 

 parts of the nucleus.) Nucleoli in early meiotic prophase nuclei of sporocytes 

 were investigated, using Carnoy's acetic alcohol for fixative, and a micro- 

 spectrographic technique combined with enzyme treatment was employed 

 for analysis of constituents. Treatment of the nucleoli with ribonuclease 

 free from deoxy ribonuclease apparently caused removal of considerable 

 material from the nucleoli, since the extinction was thereby lowered to the 

 extent of about 53%. The statement was made that about 25% of the nu- 

 cleotides (considered to be PNA nucleotides) were in the form of low-poly- 

 mer material. 



This work is illustrative of some of the experiments which have led most 

 workers to the conclusion that the nucleolus contains much PNA. However, 

 this particular investigation was concerned with plant cell nucleoli and the 

 conclusions do not necessarily apply to liver nucleoli. The results apparently 

 show the presence of PNA in the nucleoli, assuming that the ribonuclease 

 was free from protease, but, since the absorption coefficient of PNA is about 

 fifteen times greater than that of the average protein, the observed lower- 

 ing of absorption of about 53 % as the result of the action of ribonuclease 

 would indicate a nucleolar PNA content of about 7 % if it is assumed that 

 no DNA was present and that protein and PNA constituted the main bulk 

 of the nucleolar substance. However, the true PNA content might be lower 

 than this, since the fixative may have removed constituents of low molecu- 

 lar weight from the nucleoli. This investigation indicates that the nucleoli 

 of maize are largely protein, with a few per cent PNA present, and thus are 

 similar to the starfish egg nucleoli of Vincent. In regard to protein and PNA 

 content, the liver cell nucleoli isolated by Dounce et al. are also similar. 



Other histochemical studies in which reliance is made on staining reac- 

 tions alone are not so convincing. It is generally accepted that most nucleoli 

 stain pink with methyl green-pyronin, but this stain does not reliably 

 distinguish DNA from PNA but rather indicates the degree of polymeriza- 

 tion of the nucleic acid being stained, ^^^"^^^ or possibly the way in which the 



1" A. W. Pollister and C. Leuchtenberger, Nature 163, 360 (1949). 



i"C. Leuchtenberger, M. Himes, and A. W. Pollister, Anat. Record 105, 107 (1949). 



