Investigation of the repetitive sequences in calf DNA by cleavage with restriction nucleases

Abstract

Calf thymus DNA was digested with the restriction nucleases from Escherichia coli carrying resistance transfer factor I, Haemophilus influenzae Rd and Bacillus subtilis X5 (EcoRI, Hind II, and Bsu, respectively) and submitted to polyacrylamide gel electrophoresis. About 10% of the DNA migrated as discrete fragments in 8, 16, and 30 bands, respectively, superimposed upon a continuous distribution of various size DNA fragments. The fragments within the bands are repeated 2000 to 160000 times in the haploid genome. Their sizes range from about 10 to a few thousand nucleotide pairs. About 5% of the DNA in EcorRI and Hind II digests migrated in a band at the position of undigested DNA, probably due to the resistance of long stretches of DNA against these nucleases. Calf DNA fragments obtained with EcoRI and Hind II were isolated by preparative gel electrophoresis. DNA from the bands showed the behaviour of repetitive DNA in renaturation experiments. An EcoRI fragment 1300-nucleotide-pairs long, which represents 6% of the calf genome and occurs 130000 times, is tandemly repeated (derived from the satellite of 1.714 g/cm3, see below). Another EcoRI fragment of 970 nucleotide paris, which represents 0.5% of the calf genome and is derived from the DNA of 1.710 g/cm3 seems to be structurally related to the foregoing fragment since it shows a similar Hind II and Bsu cleavage pattern. It alternates with a 1550-nucleotide-pairs-long EcoRI fragment. In another series of experiments total calf DNA was separated into main-band and satellite fractions by density-gradient centrifugation and chromatography in the presence of a base-specific dye. Purified fractions were characterized by analytical ultracentrifugation and by Hind II and EcorRI digestions. From the cleavage patterns of purified fractions an assignment of the bands found with total calf DNA to satellite fractions was possible. Most fragments were derived from the components of density 1.709 and 1.710 g/cm3. The 1.714-g/cm3 satellite was cleaved into a 1300-nucleotide-pairs-ling EcorRI fragment and two Hind II fragments of 1100 and 180 nucleotide pairs. The satellites of 1.723 g/cm3 and 1.705 g/cm3 were not cleaved by either Hind II or EcoRI DNAase. On digestion of main band DNA with Bsu a 160-nucleotide-pairs-long fragment was obtained which was also observed, at a frequency of about 160000, in the Bsu digest of EcoRI fractions from total calf DNA.