DNA sequence organization in the pea genome

Abstract

The reassociation kinetics of pea (Pisum sativum L.) DNA fragments (300 nucleotides) were measured with hydroxylapatite. The most slowly reassociating fragments do so with a rate constant of 2 .times. 10-4 l mol-1 s-1, as determined from experiments with total DNA as well as with a tracer enriched for slowly renaturing sequences. This rate is about 1000 times slower than that observed for Escherichia coli DNA included as an internal kinetic standard, indicating a kinetic complexity of 4.5 .times. 109 nucleotide pairs or 4.6 pg of DNA/haploid nucleus. This estimate is in good agreement with previous chemical and cytophotometric measurements. The majority (85%) of the 300 nucleotide fragments contain repetitive sequences. While the reassociation of repetitive DNA could be modeled with 2 theoretical 2nd-order components, the data did not specify a unique solution. The reassociation kinetics of isolated high- and low-frequency fractions indicate that repetitive sequence families in pea DNA probably cover a broad range of frequencies ranging 100-10,000 or more copies/haploid genome. Single-copy sequences account for about 30% of the DNA, but because of extensive interspersion of repetitive sequences only about 15% of 300 nucleotide fragments reassociate with single-copy kinetics. Studies of hydroxylapatite binding as a function of fragment length indicate that the major class of single-copy sequences has a modal length of about 300 nucleotides. Long tracer reassociation kinetics indicate that sequences with an apparent repetition frequency of about 10,000 copies are interspersed at intervals of < 1300 nucleotides throughout 75% of the genome. At a detection limit of about 3%, no single-copy sequences longer than 1000 nucleotides are found.