For the Absence of Histone H 1 in a Mitotically Dividing

Antibodies directed against whole histone and purified lysine-rich histone H1 extracted f rom isolated macronuclei of the ciliate Tetrahymena were obtained and conjugated to fluorescein isothiocyanate. The fluorescein-antibody conjugates were used to directly label Tetrahymena cells. Both macroand micronuclei were visibly fluorescent in cells stained with anti-whole histone conjugate. However , the anti-H1 conjugate only labeled macronuclei . This in situ demonstra t ion of the lack of positive immunofluorescent staining of micronuclei with anti-H 1 conjugate provides further evidence for the absence of H1 in the genetically inactive, mitotically dividing Tetrahymena micronucleus. The lysine-rich histone, H1, has been implicated in a variety of nuclear processes. Recent evidence links H1 and its phosphorylation in a number of cell types with the alteration of the structure of interphase chromatin throughout the cell cycle (16, 18), including the conversion of nonproliferating cells to proliferating ones (17), rates of cell replication (1, 2, 28), chromosome condensation during mitosis (4, 5, 25), premitotic separation of sister chromatids (26), and the control of genetic activity (19, 24, 27). Studies done on the histones of the ciliate Tetrahymena have indicated that H1 is present, in multiple molecular species each capable of being phosphorylated, in the amitotically dividing, genetically active macronucleus of this organism (14). 1 However, the mitotically dividing, genetically inactive micronucleus apparently lacks histone H1 (12). These findings impose certain limits 1 Johmann, C. A., and M. A. Gorovsky. Manuscript in preparation. on the speculations concerning the biological role(s) of H1 in chromatin structure and function (12, 13). Since histone H1 is highly susceptible to proteolyric degradation (3, 30) and is easily dissociated from chromatin (10), the absence of H1 in isolated Tetrahymena micronuclei conceivably could be the result of a preparative artifact. Data obtained by Gorovsky and Keevert (12) argue strongly against such artifactual loss of H1 from micronuclei. However, studies done on isolated nuclei and chromatin cannot unequivocally dismiss the possibility that in vivo micronuclei contain H1. Thus, an immunofluorescence analysis was undertaken to determine the presence or absence of histone H1 in the macroand micronuclei of Tetrahymena strain BVII. Antibodies directed against BVII whole histone and purified H1 were isolated, conjugated to fluorescein isothiocyanate, and used to stain BVII cells. The results confirm our previous conclusion that micronuclei lack histone H1 (12). THE JOURNAL OF CELL BIOLOGY" VOLUME 71, 1976' pages 89-95 89 on A ril 4, 2017 D ow nladed fom Published October 1, 1976