Parseghian M H, Clark R F, Hauser L J, Dvorkin N, Harris D A, Hamkalo B A
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
Chromosome Res. 1993 Jul;1(2):127-39. doi: 10.1007/BF00710036.
Four histone H1 subtypes and H1(0) were fractionated from human placental nuclei and purified to homogeneity by a combination of Bio-Rex 70 chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Polyclonal antibodies were generated in rabbits against one of these subtypes designated H1-3. Antibodies reacted only against this subtype in enzyme-linked immunosorbent assays and Western assays; subtype specificity was documented further by Western blotting of cell and nuclear extracts. They crossreacted with monkey H1, but not with H1 from other vertebrates tested. The epitope(s) recognized were mapped by immunoblotting against peptides prepared by cleavage with N-bromosuccinimide (NBS) and alpha-chymotrypsin; it includes the variant amino-terminal tail of the protein as well as a portion of the globular domain. The antibody stains mitotic chromosomes weakly but uniformly and, unlike antibodies that recognize total H1 which show uniform nuclear staining after indirect immunofluorescence localization, anti-H1-3 exhibits preferential labelling of the nuclear periphery. This non-uniform staining suggests compartmentalization of this subtype which may have functional significance with respect to differential chromatin condensation.
从人胎盘细胞核中分离出四种组蛋白H1亚型和H1(0)，并通过Bio-Rex 70色谱法和反相高效液相色谱法（RP-HPLC）相结合的方法将其纯化至同质。用兔制备了针对其中一种命名为H1-3的亚型的多克隆抗体。在酶联免疫吸附测定和蛋白质免疫印迹分析中，抗体仅与该亚型发生反应；通过对细胞和核提取物进行蛋白质免疫印迹进一步证明了亚型特异性。它们与猴H1发生交叉反应，但与所测试的其他脊椎动物的H1不发生交叉反应。通过针对用N-溴代琥珀酰亚胺（NBS）和α-胰凝乳蛋白酶切割制备的肽进行免疫印迹来定位所识别的表位；它包括蛋白质的可变氨基末端尾巴以及部分球状结构域。该抗体对有丝分裂染色体的染色较弱但均匀，并且与识别总H1的抗体不同，后者在间接免疫荧光定位后显示均匀的核染色，抗H1-3表现出对核周边的优先标记。这种不均匀染色表明该亚型的区室化，这可能在差异染色质凝聚方面具有功能意义。
