Konkel D A, Ingram V M
Nucleic Acids Res. 1978 Apr;5(4):1237-52. doi: 10.1093/nar/5.4.1237.
Hg-UMP-containing transcripts made from chick erythroid chromatins with E. coli RNA polymerase hybridize to chick globin cDNA. Contamination with endogenous globin RNA has been largely removed by purification on SH-agarose columns at 55 degrees C. Some endogenous globin mRNA sequences remain, probably as hybrids with "anti-sense" Hg-transcripts produced by RNA-dependent RNA synthesis. Heating to 115 degrees C before SH-agarose chromatography eliminates these contaminants. Hg-transcripts from adult and embryonic erythroid chromatins purified by this method are hybridized to globin cDNA; they contain a 4- to 6-fold higher proportion of globin-specific sequences (10-13 PPM) than do transcripts from brain chromatin. Dissociation of erythroid chromatins in salt and urea, followed by reconstitution using standard methods, destroys even this low degree of specificity.
用大肠杆菌RNA聚合酶从鸡红细胞染色质制备的含汞尿苷酸转录本与鸡珠蛋白cDNA杂交。通过在55℃下于SH -琼脂糖柱上纯化,已基本去除了内源性珠蛋白RNA的污染。仍保留一些内源性珠蛋白mRNA序列,可能是与由RNA依赖性RNA合成产生的“反义”汞转录本形成的杂交体。在SH -琼脂糖柱层析之前加热到115℃可消除这些污染物。用这种方法纯化的成年和胚胎红细胞染色质的汞转录本与珠蛋白cDNA杂交;它们所含的珠蛋白特异性序列比例(10 - 13 PPM)比脑染色质的转录本高4至6倍。红细胞染色质在盐和尿素中解离,然后用标准方法重构,甚至会破坏这种低程度的特异性。