204 NORBERT SCHCMMELFEDER 



of the pyknotic nuclei was observed. This result corresponds to that of 

 Sparrow et al. (1952) on Trillium nuclei. 



Analogous to Burstone's (1953) observations, Kaufmann et al. (1955) 

 have shown in experiments on meristematic cells of onion roots that DNA 

 in irradiated cells is more easily dissoKed by DNase than in nonirradiated 

 controls. In contrast, similar experiments on grasshopper embryos showed a 

 higher resistance of the irradiated nuclei, i.e., by their DNA. to enzymatic 

 hydrolysis. These different results stimulated us to seek information on 

 whether the de\elopment of pyknosis of granule cell nuclei following ir- 

 radiation alters their response to depolymerizing and hydrolyzing agents. 



The results of these experiments showed that pyknotic nuclei of granule 

 cells are more resistant than nonpyknotic nuclei to treatment with depoly- 

 merizing agents, e.g. to boiling water or to hydrochloric or perchloric acid. 

 Pro\ided the reaction conditions are favorable, only in the nonirradiated 

 nuclei did exposure to these agents result in depolymerization. These nuclei 

 showed red fluorescence after staining with buffered solutions of acridine 

 orange, pH 5.0 to 7.0, whereas the pyknotic nuclei still fluoresced bright 

 yellow owing to retained high polymerization of DNA. The difference in 

 color and intensity ot the fluorescence was so conspicuous that each indi- 

 vidual pyknotic nucleus could easily be obser\ed. 



Methyl green, which stains only highly polymerized DNA, has yielded 

 the same results in companion sections. The pyknotic nuclei were still stain- 

 able with methyl green, whereas unaltered granule cell nuclei could not 

 be stained after pretreatment with depolymerizing agents. 



Further experiments showed that enzymatic breakdown of DNA, using 

 DNase, occurs more slowly in pyknotic than in intact nuclei. This result is 

 similar to that ob.served by Kaufmann ct al. (1955) in irradiated grass- 

 hopper embryos. 



Hydrolysis with hydrochloric or perchloric acid removed DNA from the 

 nuclei of unaltered granule cells, whereas DNA of the pyknotic nuclei in 

 irradiated granule cells was only slightly depolymerized. Using favorable 

 conditions of hydrolyzation. it is easy to demonstrate selectively the pyk- 

 notic nuclei after staining with acridine orange, whereas nonpyknotic nuclei, 

 which are not altered by the irradiation and which are deprived of the 

 DNA by the foregoing hydrolysis, remain imstained. 



In our experimental study we were unable to determine whether increased 

 resistance of the irradiated pyknotic nuclei to DNase and to depolymerizing 

 and hydrolysing chemical agents is an immediate and specific effect due to 

 the action of ionizing radiation on the DNA of the nuclei. The observations 

 of Kaufmann ct al. (1955) have indicated that the structural organization 

 of the nucleoproteins in irradiated nuclei is changed. On the other hand 

 Yakar (1952) has demonstrated in plant cells that the speed of enzymatic 



