Stargell L A, Bowen J, Dadd C A, Dedon P C, Davis M, Cook R G, Allis C D, Gorovsky M A
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Genes Dev. 1993 Dec;7(12B):2641-51. doi: 10.1101/gad.7.12b.2641.
Vegetative cells of the ciliated protozoan Tetrahymena thermophila contain a transcriptionally active macronucleus and a transcriptionally inactive micronucleus. Although structurally and functionally dissimilar, these nuclei are products of a single postzygotic division during conjugation, the sexual phase of the life cycle. Immunocytochemical analyses during growth, starvation, and conjugation were used to examine the nuclear deposition of hv1, a histone H2A variant that is found in macronuclei and thought to play a role in transcriptionally active chromatin. Polyclonal antisera were generated using whole hv1 protein and synthetic peptides from the amino and carboxyl domains of hv1. The transcriptionally active macronuclei stained at all stages of the life cycle. Micronuclei did not stain during growth or starvation but stained with two of the sera during early stages of conjugation, preceding the stage when micronuclei become transcriptionally active. Immunoblot analyses of fractionated macro- and micronuclei confirmed the micronuclear acquisition of hv1 early in conjugation. hv1 staining disappeared from developing micronuclei late in conjugation. Interestingly, the carboxy-peptide antiserum stained micronuclei only briefly, late in development. The detection of the previously sequestered carboxyl terminus of hv1 may be related to the elimination of hv1 during the dynamic restructing of micronuclear chromatin that occurs as the micronucleus enters a transcriptionally incompetent state that is maintained during vegetative growth. These studies demonstrate that the transcriptional differences between macro- and micronuclei are associated with the loss of a chromatin component from developing micronuclei rather than its de novo appearance in developing macronuclei and argue that hv1 functions in establishing a transcriptionally competent state of chromatin.
嗜热四膜虫的纤毛原生动物的营养细胞含有一个转录活跃的大核和一个转录不活跃的小核。尽管这些细胞核在结构和功能上不同，但它们是生命周期有性阶段即接合过程中单个合子后分裂的产物。利用生长、饥饿和接合过程中的免疫细胞化学分析来检测hv1的核沉积情况，hv1是一种组蛋白H2A变体，存在于大核中，被认为在转录活跃的染色质中发挥作用。使用完整的hv1蛋白以及来自hv1氨基和羧基结构域的合成肽产生多克隆抗血清。转录活跃的大核在生命周期的所有阶段都有染色。小核在生长或饥饿期间不染色，但在接合早期，在小核变得转录活跃之前，用两种血清染色。对分离的大核和小核进行免疫印迹分析证实了在接合早期小核获得了hv1。在接合后期，hv1染色从发育中的小核中消失。有趣的是，羧基肽抗血清仅在发育后期短暂地对小核进行染色。检测到先前被隔离的hv1羧基末端可能与小核染色质动态重组过程中hv1的消除有关，这种重组发生在小核进入转录无活性状态时，该状态在营养生长期间得以维持。这些研究表明，大核和小核之间的转录差异与发育中的小核中染色质成分的丧失有关，而不是其在发育中的大核中重新出现，并表明hv1在建立染色质的转录活性状态中起作用。
