Reassociation kinetics of non-histone-bound DNA sites.

Functional significance of rat liver nonhistone protein-DNA interactions: RNA hybridization of protein-bound DNA

“…The DNA can be isolated, repurified, and sheared to 350 nucleotide pairs in a French pressure cell at 20000 psi (66% glycerol, 0.1 M sodium acetate). The selfreassociation of these fragments indicates that two major reassociating components are present (Jagodzinski et al, 1979) (Figure 4). The fast component reassociated with a C0t 1/2 = 0.01 M s and the slow component with a C70í 1/2 = 256 M s. The slow component was isolated by reassociating 3Hlabeled, sheared, bound DNA to a C0t = 1000.0 M s, isolating the double-stranded DNA, and reassociating this DNA again to a C0t -1.0 M. The single-stranded DNA was then col-LOG EQUIVALENT m R"t figure 6: Hybridization of single-copy bound DNA with liver and kidney polysomal mRNA.…”

“…From purified nuclei of rat liver, a group of DNA-binding NHP can be extracted by selective salt solubilization (Sevall et al, 1975;Rastraba & Wang, 1976). The DNA bound by these nonhistone proteins can be isolated, and analysis of its reassociation kinetics indicates that it is a subset of repetitive and single-copy sequences (Jagodzinski et al, 1979). The single-copy component of the bound DNA is 10% of the complexity of the whole single-copy component of the rat genome and has been used as a tracer in RNA-driven hybridizations.…”

“…Isolation of Chromosomal Components. The purification of rat liver NHP has been described previously (Jagodzinski et al, 1979). Nonhistone proteins eluted between 0.35 and 1.0 M sodium chloride from purified nuclei represent 3.7% of the nuclear protein, and when mixed at ratios of 3:1 with 3400 nucleotide pair DNA (1 ¿ig/mL), 0.24 M ionic strength (pH 8.0), 5.0% of the DNA is retained as protein-bound DNA.…”

“…With increased DNA fragment length, the nonhistone protein-bound DNA is enriched with a subset of single-copy DNA sequences which neighbor the repetitive protein-binding site. This subset of sequences is enriched in transcribed single-copy sequences in rat liver mRNA (Jagodzinski et al, 1979).…”