RAMAN MICROSPECTROSCOPIC STUDY OF LOW-PH-INDUCED CHANGES IN DNA-STRUCTURE OF POLYTENE CHROMOSOMES

The effects of low-pH treatments on DNA structure in polytene chromoso mes of Chironomus thummi thummi have been studied by Raman microspectr oscopy. Measurements were carried out on chromosomes at low pH and on chromosomes reneutralized after a short exposure to low pH. Protonatio n of adenine residues and subsequent unpairing of adenine (A) and thym ine (T) were found to commence already above pH 3.6 and are completed at pH 2.2. Protonation of the cytosine-guanine base pair also starts a bove pH 3.6. It leads to an intermediate protonated, most likely Hoogs teen-type, guanine-cytosine base pair. Unpairing of G and C residues s tarts between pH 2.6 and 2.2 and continues below pH 2.2. Brief treatme nt of chromosomes at a pH less than or equal to 2.2, i.e., at a pH whe re both AT and GC base pairs are disrupted, was found to lead to irrev ersible changes in DNA conformation upon return to neutral pH. These w ere most pronounced after treatment with 45% acetic acid. More than 10 % of the A and T residues was found to have adopted a non-B-DNA confor mation. Evidence was found for denaturation of the B-form backbone. Th e amount of protein extracted from the chromosomes was strongly pH-dep endent. Treatment at pH 3.6 did not cause noticeable protein extractio n, while treatment with 45% acetic acid extracted more than 50% (by we ight) of the chromosomal proteins.