64 HEWSON SWIFT 



quantitative estimates has been suggested,*^ although in some cases non- 

 specific staining may occur. ^^ The binding of dyes mordanted with heavy 

 metal ions to produce dye lakes is extremely strong and will take place at 

 very low pH levels (e.g., 0.8 for gallocyanine).^' Further investigation on 

 the stoichiometry of such reactions would be of interest. 



After treatment with deoxyribonuclease, the nuclei in many tissues do 

 not stain at all with basic dyes, except for the nucleoli. Ultraviolet measure- 

 ments on onion and lily nuclei, however,^" -^^ indicate the presence of a 

 considerable amount of material with a nucleic acid absorption curve that 

 is removable with ribonuclease. It seems likely that this material repre- 

 sents PNA, where the phosphoryl groups are not available to basic dye 

 binding, possibly because of protein blocking. 



Because of the variables mentioned here, it seems that certain differences 

 that have been reported in dye binding between tissues should be inter- 

 preted with some caution. Any "increase" in nucleic acid with mitosis 

 needs to be investigated with other independent techniques such as ultra- 

 violet absorption. Also the extent of "linkage" between, for example, DNA 

 and histone probably cannot be estimated accurately by determining the 

 increase in basic dye binding caused by histone removal. Histone removal 

 would be expected to shift the equilibrium in the staining solution toward 

 increased binding, but the increase produced would be of different magni- 

 tude depending on dye pH, concentration, etc. Kaufmann, McDonald, and 

 Gay** reported that methyl green staining of onion root nuclei could be 

 aboUshed, under certain conditions, either by deoxyribonuclease or ribo- 

 nuclease. Rather than concluding that PNA is essential to DNA polymer- 

 ization, a plausible explanation Avould seem to be that the amino groups 

 known to be uncovered by PNA removal are sufficient in some cases suc- 

 cessfully to inhibit methyl green binding by the DNA. 



In conclusion it should be emphasized that, for a qualitative picture of 

 nucleic acid distribution in tissues, dye binding may be a valuable research 

 tool. If used properly basic dyes are practically specific for nucleic acid, 

 and under special conditions can differentiate between DNA and PNA. 

 The staining of tissues, at least as usually carried out, is subject to too many 

 variables, however, to provide stoichiometric binding to nucleic acid, and 

 thus dye content, as measured by photometric instruments, cannot be 

 relied upon to give an accurate estimate of the amount of nucleic acid 

 present. In certain cases measurements of dye bound may provide accurate 



61 L. Einarson, Ada Pathol. Scand. 28, 82 (1951). 

 «U. Stenram, Exptl. Cell Research 4, 383 (1953). 

 " H. Swift, Texas Repts. Biol, and Med. 11, 755 (1953). 



" B. P. Kaufmann, M. R. McDonald, and H. Gay, J. Cellular Comp. Physiol. 38, 

 Suppl. 1,71 (1951). 



