A CHROMATOGRAPHIC STUDY OF HYDROLYSIS IN THE FEULGEN NUCLEAL REACTION

Abstract

Anthers from flower buds of Lilium longiflorum were frozen, dried, fixed in warm 75% alcohol, and sectioned in paraffin. The sections were then placed in a glass tube with a fritted glass filter disc fused to the lower end. By dipping the filter end of the tube into vials of reagents it was possible to process free sections in a manner similar to the standard cytological procedures commonly used in carrying out the Feulgen reaction. The several acid hydrolysates (corresponding to different hydrolysis times) were then analyzed chromatographically for their thymine, uracil, and adenine content. By modifying the procedure slightly it was also possible to determine whether these bases occurred free or as derivatives. Hydrolysis of tissue was accomplished by 10% perchloric acid at 20[degree]C. From similarly treated sections mounted on microscope slides and stained with Feulgen reagent maximum staining was shown to occur at 19 hours. From microphotometric measurements it was also shown that this amount differed insignificantly from the amount in nuclei fixed and hydrolyzed by more conventional methods. Uracil-containing material was almost completely separated from thymine-containing material of tissue sections by 1 1/2 hours. Adenine, as the base, was all removed by 19 hours. Detectable amounts of thymine-containing material appeared in the hydrolysate shortly after the beginning of hydrolysis and by optimum hydrolysis approximately 2/3 of the total thymine of the tissue was lost. Removal of these thymine-containing fragments was linear with respect to time during the first 24 hours and occurred at a relatively high rate. Removal after 24 hours also appeared to be linear but at a markedly lower rate. These findings were discussed in connection with the use of the Feulgen method as a means of determining relative amounts of deoxyribonucleic acid in nuclei from measurements on amount of Feulgen dye.