THE MITOTIC CYCLE 



or even recognize particular constituents within an area of a complex 

 mixture of the order of a few square microns goes beyond the exactitude 

 demanded of any analytical procedure elsewhere in microchemistry. 

 However, rather than sit with folded hands until fully adequate methods 

 are available, students of cellular biology have chosen to make use of the 

 present inadequate procedures although the results obtained have 

 often only a provisional status. To allow the tentative generalizations 

 which emerge to harden into dogma by frequent reiteration is neverthe- 

 less an avoidable impediment to the development of the subject. 

 Furthermore, it must be remembered that much of our knowledge of 

 intracellular physiology is still well within the early phase of the 

 development of a science at the qualitative level. 



With these considerations in mind we must now turn to the discussion 

 of two major developments in research relating to the cytochemical 

 localization of the nucleic acids, namely the development of ultra- 

 violet microspectrometry by Caspersson and his school, and secondly 

 the isolation of separate nucleoclastic enzymes in approximate states 

 of purity, exclusively affecting the two types of nucleic acid. 



NUCLEOCLASTIC ENZYMES 



It will be convenient here to discuss first the latter of these two develop- 

 ments. Following the usual nomenclature which uses the name of the 

 appropriate substrate, these enzymes are now known as ribonuclease 

 and deoxyribonuclease respectively. Both have been prepared from the 

 pancreas, by which they are secreted for the digestion of nucleic acids 

 liberated by the stomach acid from dietary nucleoproteins. The intra- 

 cellular nucleases in other tissues are probably not identical with these 

 pancreatic enzymes. 



Ribonuclease proved much the easier enzyme to prepare, thanks to 

 its remarkable stability towards both temperature and pH (Jones,*^ 

 DuBOS and Thompson*^). It was crystalHzed first by Kunitz^^ in 1940, 

 and found to be an albumen-like protein, with a molecular weight of 

 approximately 15,000. 



The method described by Kunitz and Northrop ^^ for the separation 

 of chymo-trypsin from the pancreas was found to be of service also for 

 the nucleases. The tissue is extracted in cold o-25N sulphuric acid and by 

 this means the activation of the tryptic enzymes is prevented. Ribonu- 

 clease is resistant to trypsin, but deoxyribonuclease is readily attacked 

 thereby. From the filtered acid extract, the various enzyme proteins 

 can be fractionally precipitated with diflferent concentrations of 

 ammonium sulphate; first deoxyribonuclease at a saturation of 0-4 and 

 ribonuclease between o-6 and o-8. Deoxyribonuclease was prepared 

 in this way by Fischer et alii^^ in 1941, and by McCarty" in 1946. 



