Aggregate formation from short fragments of plant DNA

Abstract

Large aggregates have been observed after partial reassociation of pea (Pisum sativum L.) DNA preparations sheared to mean single strand fragment lengths as short as 350 nucleotides. At high DNA concentrations and conditions of salt and temperature which require only moderate precision of base pairing, aggregates pelletable by brief centrifugation account for 30 to 40% of the total DNA from peas, while calf thymus DNA reassociated under similar conditions forms less than 10% pelletable structures. In contrast to networks formed during the reassociation of long DNA fragments containing interspersed repetitive sequences, these aggregates contain a high percentage of double-stranded DNA and are enriched in repetitive sequences.Aggregates detectable by centrifugation do not begin to appear until after extensive repetitive sequence reassociation has already occurred. The results are consistent with a model involving secondary reassociation between single-stranded regions ("hanging tails") remaining after initial duplex formation. This process would lead to formation of large multimers of the original fragments, analogous to the large hyperpolymers which have been observed in extensively reassociated prokaryotic DNA. Randomly sheared fragments containing short (about 300 base pairs) repetitive sequences interspersed with single copy DNA would not be expected to hyperpolymerize significantly under these conditions. I suggest, as a working hypothesis, that much of the repetitive sequence DNA in peas is contained in regions considerably longer than 300 base pairs.