On the Denaturation of Chromosomal Dna in Situ

Denaturation studies on extracted DNA in vitro have made major contributions toward understanding the physical make-up of the molecule, its possible behavior in replication, and its response to various modifying agents, such as irradiation and incorporated base analogues.The present report deals with the effect of denaturing conditions on DNA in chromosomes prepared for microscopic observation, the visualization of the native and denatured states with the fluorochrome acridine orange, and preliminary consideration of the involvement of other chromosomal constituents in the process of denaturation.Materials and Methods.-Thetissues used in these investigations were larval salivary glands of Drosophila melanogaster.These were dissected in modifiedRinger solution' and placed on gelatinized slides in -a drop of 45 per cent acetic acid.In one experiment acid fixation was preceded by a 5-min treatment in 4 per cent neutral formaldehyde.The fixed glands were covered, squashed, and, after removal of the coverslip by freezing in liquid nitrogen, postfixed in ethanol-acetic acid (3:1), then passed through absolute and 95 per cent ethanol into 75 per cent ethanol for storage.Prior to subsequent treatments in aqueous solutions, the slides were hydrated gradually.Enzymatic digestions were carried out over periods of 3 hr at 380C.Desoxyribonuclease (Worthington), in 4 X 10-3 M MgSO4, or ribonuclease (Worthington or N.B.C.) were used at a concentration of 0.3 mg/ml.The pH was adjusted to 7.0 with NaOH for DNase and Na2HPO4 for RNase.RNase solutions were freed of possible DNase activity by heating for 20 min in boiling water.After preliminary treatments, slides were returned to distilled water, washed in acetate buffer (0.1 M, pH 4.5), and stained for 30 min in acridine orange (Harleco, 0.25 mg/ml in pH 4.5 acetate buffer).Before viewing, the preparations were washed several times in buffer, air-dried, covered with a fresh drop of buffer and a coverslip, and sealed with "Laktoseal" (Gurr).Observations of fluorescence pat- terns were made with Xenon illumination, a BG-12 exciter filter (4 mm, peak trans- mission at 4000 A), and a Leitz "blue-absorbing" barrier filter.Results and Interpretation.-Theresults obtained in terms of secondary fluores- cence colors, are listed in Tables 1-3.