Potashkin J A, Huberman J A
Exp Cell Res. 1986 Jul;165(1):29-40. doi: 10.1016/0014-4827(86)90530-6.
We have used two different approaches to determine whether particular DNA sequences are specifically associated with high-salt-treated residual nuclei of Saccharomyces cerevisiae. First, libraries of yeast DNA in phage lambda were probed with nick-translated total nuclear or residual nuclear DNA from unsynchronized yeast cells. None of the plaques gave a significantly stronger or weaker signal with the residual nuclear probe than with the total nuclear probe. Second, DNA was purified from whole nuclei or residual nuclei which had been isolated from cells in G1, G1/S, early S, or nuclear division. This DNA was "dot-blotted" and then probed with specific yeast DNA sequences. Ribosomal DNA was 2- to 3-fold enriched in residual nuclei in late G1, G1/S, and early S, and 2 microns plasmid DNA sequences were 3- to 5-fold depleted during nuclear division and early G1. However, ARS1, TRP1, CEN6, and a telomere sequence were neither enriched nor depleted at any time during the cell cycle.
我们采用了两种不同的方法来确定特定的DNA序列是否与经高盐处理的酿酒酵母残余细胞核有特异性关联。首先，用来自未同步化酵母细胞的经切口平移的全核或残余核DNA探测λ噬菌体中的酵母DNA文库。与全核探针相比，没有一个噬菌斑在用残余核探针时给出明显更强或更弱的信号。其次，从处于G1期、G1/S期、早S期或核分裂期的细胞中分离得到的全核或残余核中纯化DNA。将该DNA进行“点杂交”，然后用特定的酵母DNA序列进行探测。核糖体DNA在G1晚期、G1/S期和早S期的残余核中富集了2至3倍，而2μm质粒DNA序列在核分裂期和G1早期减少了3至5倍。然而，ARS1、TRP1、CEN6和端粒序列在细胞周期的任何时候既没有富集也没有减少。
