Detection of long eukaryote-specific pyrimidine runs in repetitive DNA sequences and their relation to single-stranded regions in DNA isolated from sea urchin embryos.

Abstract

Polypyrimidines, 50 to 200 nucleotides in length, have been found in the nuclear DNA of Strongylocentrotus purpuratus embryos. The distribution of lengths of pyrimidine runs resulting from depurination of main-density band sea urchin DNA, separated by CsCl gradient centrifugation, is the same as that obtained from the unfractionated DNA. When embryo DNA is separated into unique and repetitive sequence fractions, the polypyrimidines having more than 50 nucleotides are associated, for the most part, with the repetitive sequence fractions of the DNA. Removal of regularly spaced, naturally occurring, single-stranded regions (by digestion with Aspergillus oryzae S1 nuclease) from morula stage native DNA causes a partial depletion of polypyrimidines 60 to 80 nucleotides in length. Treatment of gastrula stage native DNA, which lacks single-stranded regions, with S1 nuclease does not alter the size pattern of polypyrimidines, compared with untreated embryo DNA. Removal of single-stranded regions from repetitive sequence morula stage DNA by treatment with S1 nuclease also causes a reduction in polypyrimidines 60 to 80 nucleotides in length. It is concluded that eukaryote-specific polypyrimidines, 50 to 200 nucleotides in length, are associated primarily with repetitive sequence DNA and that a portion of the isostichs (see Introduction) 60 to 80 nucleotides in length occur at nonrandom sites which are single-stranded during pre-gastrula stage development. This latter conclusion, and a previous finding that these single-stranded regions occur as staggered pairs (on opposite strands) at the center of native unit length sections of duplex morula stage DNA, suggests that the 60 to 80 nucleotide-length polypyrimidines may be in register with sites which demarcate bidirectional replication regions along the genome.