Hough C J, Smulson M E
Biochemistry. 1984 Oct 9;23(21):5016-23. doi: 10.1021/bi00316a029.
We have investigated whether transcriptionally active or inactive gene sequences are associated in vivo with poly(adenosine diphosphate ribosylated) regions of chromatin. Soluble HeLa cell chromatin derived from nuclei treated either briefly or extensively with micrococcal nuclease was fractionated on an anti-poly(adenosine diphosphate ribose)-Sepharose column [Malik, N., Miwa, M., Sugimura, T., Thraves, P., & Smulson, M. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2554-2558] to obtain fractions that were enriched or depleted in poly(ADP-ribosylated) chromatin. DNA obtained from these fractions was then probed for active and inactive gene sequences with a cDNA probe made from total cell mRNA and a probe for the beta-globin gene. Chromatin enriched in poly(ADP-ribosylated) nucleosomes contained both active and inactive gene sequences as detected by the probes and appeared to be more nuclease sensitive than that found in the fraction of chromatin depleted of poly(ADP-Rib). Poly(ADP-ribosylated) chromatin from nuclei digested briefly with nuclease showed an enrichment in both active and inactive genes while that treated extensively with nuclease showed either no enrichment or a depletion of active and inactive genes. Actively transcribed chromatin was digested at a rate several times that of the bulk or inactive chromatin. Nevertheless, the enrichment of active genes in poly(ADP-ribosylated) nucleosomes derived from brief nuclease digestion was greater than that of inactive genes. These results are interpreted as showing that some, but not all, of actively transcribed chromatin contains associated poly(ADP-ribosylated) proteins. However, since poly(ADP-ribosylated) proteins are also associated with inactive genes, the function of this modification cannot be assigned solely to transcription.
我们研究了转录活性或非活性基因序列在体内是否与染色质的聚（二磷酸腺苷核糖基化）区域相关联。用微球菌核酸酶短暂或长时间处理细胞核后得到的可溶性海拉细胞染色质，在抗聚（二磷酸腺苷核糖）琼脂糖柱上进行分级分离[马利克，N.，三泽，M.，杉村，T.，思雷夫斯，P.，& 斯穆尔森，M. E.（1983年）《美国国家科学院院刊》80，2554 - 2558]，以获得聚（ADP - 核糖基化）染色质富集或耗尽的级分。然后用由总细胞mRNA制备的cDNA探针和β - 珠蛋白基因探针，对从这些级分中获得的DNA进行活性和非活性基因序列检测。如探针所检测到的，富含聚（ADP - 核糖基化）核小体的染色质同时含有活性和非活性基因序列，并且似乎比聚（ADP - 核糖）耗尽的染色质级分中的染色质对核酸酶更敏感。用核酸酶短暂消化细胞核得到的聚（ADP - 核糖基化）染色质，在活性和非活性基因中均显示出富集，而用核酸酶长时间处理的染色质则要么没有富集，要么活性和非活性基因均出现耗尽。活跃转录的染色质的消化速率是整体或非活性染色质的几倍。然而，来自短暂核酸酶消化的聚（ADP - 核糖基化）核小体中活性基因的富集程度大于非活性基因。这些结果被解释为表明部分但并非全部活跃转录的染色质含有相关的聚（ADP - 核糖基化）蛋白质。然而，由于聚（ADP - 核糖基化）蛋白质也与非活性基因相关联，这种修饰的功能不能仅归因于转录。
