Takami Y, Nakayama T
Department of Biochemistry, Miyazaki Medical College, Kiyotake, Japan.
Genes Cells. 1997 Nov;2(11):711-23. doi: 10.1046/j.1365-2443.1997.1550353.x.
There is general agreement that large numbers of histone H1 are necessary for maintenance of the higher order structure of chromatin in higher eukaryotes. The chicken H1 gene family comprises six members per haploid genome, the total copy number being 12, and they encode six H1 variants which are considerably different from each other in amino acid sequence. We recently established that in two chicken DT40 mutants (1/2delta110kb and delta57kb), which lack, respectively, one allele of the gene cluster of 110 kb carrying six H1 genes, plus 33 core histone genes, and two copies each of four of the six H1 genes included in an approximately 57 kb segment of the cluster, expression of the remaining H1 genes is increased, resulting in constant steady-state levels of total H1 mRNAs. These results gave rise to the simple questions of how many H1 genes and how many H1 variants, at minimum, are necessary for the viability of DT40 cells.
We generated two DT40 mutants, delta10/12H1 and delta11/12H1, which are devoid, respectively, of two copies each of five H1 genes, and those plus a single copy of the last H1 gene, in addition to 17 core histone genes. Analyses involving a RNase protection assay, SDS-PAGE and acid-urea-PAGE revealed, not only that in the delta10/12H1 mutant the steady-state levels of total H1 mRNAs and the amounts of histone H1 were not changed, but also that in the delta11/12H1 mutant both were approximately one-half the normal levels, and the amounts of HMG proteins were increased about twofold. No alteration in the growth rate or global chromatin structure was observed in either mutant. On the other hand, the protein patterns on 2D-PAGE of the delta11/12H1 mutant were definitely distinct from those of the wild-type cell line.
These results indicate not only that a lack of five of the six H1 variants causes changes in the protein patterns, but also that only a single copy of the H1 genes is enough for cell proliferation.
人们普遍认为，在高等真核生物中，大量的组蛋白H1对于维持染色质的高级结构是必需的。鸡的H1基因家族每个单倍体基因组包含六个成员，总拷贝数为12个，它们编码六种H1变体，这些变体在氨基酸序列上彼此有很大差异。我们最近发现，在两个鸡DT40突变体（1/2delta110kb和delta57kb）中，前者分别缺失携带六个H1基因以及33个核心组蛋白基因的110 kb基因簇的一个等位基因，后者缺失该基因簇约57 kb片段中六个H1基因中的四个基因的两个拷贝，其余H1基因的表达增加，导致总H1 mRNA的稳态水平保持恒定。这些结果引发了一些简单的问题，即DT40细胞的存活至少需要多少个H1基因和多少种H1变体。
我们构建了两个DT40突变体，delta10/12H1和delta11/12H1，它们分别缺失五个H1基因的两个拷贝，以及除17个核心组蛋白基因外的最后一个H1基因的一个拷贝。涉及核糖核酸酶保护分析、SDS-PAGE和酸性尿素-PAGE的分析表明，不仅在delta10/12H1突变体中总H1 mRNA的稳态水平和组蛋白H1的量没有变化，而且在delta11/12H1突变体中两者均约为正常水平的一半，并且HMG蛋白的量增加了约两倍。在这两个突变体中均未观察到生长速率或整体染色质结构的改变。另一方面，delta11/12H1突变体的二维电泳蛋白质图谱与野生型细胞系的图谱明显不同。
这些结果不仅表明六种H1变体中的五种缺失会导致蛋白质图谱发生变化，而且表明H1基因的单个拷贝就足以支持细胞增殖。
