Reassociation characteristics of DNA synthesized in wounded pea seedlings

Abstract

Summary A reassociation study was carried out with DNA isolated from unwounded and wounded pea epicotyl tissues (sheared DNA, mean molecular weight 450,000 d). Spectrophotometric reassociation curves of reference DNA's, of control and wounded tissue DNA, were identical up to C0t 100. In order to compare the reassociation characteristics of newly synthesized DNA, labelled by radioactive precursor incorporation, with the reassociation characteristics of total isolated DNA, the reassociation mixture was fractionated by hydroxylapatite column chromatography. By this technique it was shown that the reassociation of newly synthesized and total DNA coincides during the initial phase (up to C0t 0.1). However, between C0t 0.1 and 1000 labelled DNA reassociated markedly slower than total DNA; a difference of 10 to 15% was found. Reassociation was also studied in the presence of an excess of driver DNA, originating from tissues with a varying degree of polyploidy: a similar difference was stated between the reassociation characteristics of total and labelled DNA. This peculiar reassociation is not due to insufficient procedures but represents a real phenomenon, resulting from the underreplication or non-replication of repetitive DNA sequences in cambium nuclei. The main characteristics of reassociated duplexes (density, thermal stability and fragment-length) prove that the reassociation is a reliable reaction. Our results are mainly discussed in relation to the thesis that differential DNA replication is involved in stress-induced DNA synthesis.