Release of a globin gene enriched chromatin fraction from chicken erythrocyte nuclei following DNase II digestion

Mild digestion of chicken erythrocyte nuclei with deoxyribonuclease II results in the release of a chromatin fraction which is 4- to 13-fold enriched for the globin coding sequences when compared to total chicken DNA. The remaining nuclear pellet is depleted in these sequences. A maximum of 25% of the globin genes have been recovered in the released fraction. The addition of 5 mM sodium butyrate to the digestion buffer is required to obtain reproducible globin gene enrichment. The released fraction contains equimolar amounts of the four core histones and a subset of the nonhistone chromosomal proteins. The globin genes are released as large chromatin fragments which exceed the 1.6 kilobase size of the transcribed portion of the gene.     

Reconstitution of endogenous DNA synthesis in isolated nuclei and template activity of chromatin with respect to mode of inhibition by aphidicolin

We succeeded in reconstituting the endogenous nuclear DNA synthesis of the sea urchin. Endogenous DNA synthesis of isolated nuclei was reconstituted by mixing the salt-treated nuclei (chromatin exhibiting essentially no endogenous DNA synthesis) and the salt extract containing DNA polymerase-alpha. DNA synthesis in this reconstitution system showed a level of activity and a mode of inhibition by aphidicolin similar to those of the original isolated nuclei (noncompetitive with respect to dCTP). On the other hand, the inhibitory mode was competitive with respect to dCTP in DNA synthesis in the reconstituted system obtained from the chromatin and purified DNA polymerase-alpha, indicating that some other factor(s) in addition to DNA polymerase-alpha is necessary for the reconstitution with reference to the inhibitory mode of aphidicolin. We also studied the template activity of the chromatin. When chromatin was used as a template, inhibition by aphidicolin of DNA polymerase-alpha was noncompetitive and uncompetitive with respect to the template at high and low concentrations, respectively. Treatment of chromatin with 5 M urea gave urea-treated chromatin (nonhistone protein-deprived chromatin) and the extract (mainly nonhistone protein fraction). Inhibition by aphidicolin of DNA polymerase-alpha was uncompetitive with respect to the urea-treated chromatin. However, when chromatin reconstituted from the urea-treated chromatin and the extract was used as a template, the inhibitory mode by aphidicolin was similar to that with original chromatin, indicating that the nonhistone protein fraction contained factor(s) which modified the inhibitory mode of aphidicolin. Thus, the inhibitory mode of aphidicolin is a useful parameter for monitoring the resolution and reconstitution of endogenous DNA synthesis of isolated nuclei.     

Distribution of nucleosomes on reconstituted chromatin from cloned mouse beta-globin DNA

Relative abundance of nucleosomes on reconstituted chromatin was estimated with cloned mouse beta-globin gene DNA. Mononucleosomal DNA was isolated from reconstituted chromatin after digestion with micrococcal nuclease, nick-translated and used as a probe for blot hybridization. DNA fragments of restriction nuclease-digested globin DNA were transferred to DBM-paper and hybridized with mononucleosomal [32P] DNA probe. The results showed non-random distribution of nucleosomes.