Timing of the appearance of macronuclear-specific histone variant hv1 and gene expression in developing new macronuclei of Tetrahymena thermophila.

Abstract

Vegetative cells of the ciliated protozoan T. thermophila contain a transcriptionally active macronucleus and a transcriptionally inactive micronucleus. Earlier studies demonstrated the existence of a macronuclear-specific histone variant, hv1, which is enriched in small punctate regions in nucleoli of several mammalian cell lines. Evidently this histone variant is highly conserved in evolution and may be associated with actively transcribed sequences. Despite large differences in structure and function during vegetative growth, macro- and micronuclei are related. During conjugation, the sexual phase of the life cycle in Tetrahymena, postzygotic division products of micronuclei give rise to new micro- and macronuclei, while the old macronucleus moves to the posterior of each cell and is eliminated. Using antiserum specific for hv1, determined by indirect immunofluorescence the time during conjugation at which hv1 first appears in the developing new macronuclei. In growing, starved and young mating cells (2-5 h after mixing opposite mating types), only macronuclei are detected with affinity-purified antibodies against hy1. Newly formed macronuclei are either not stained or only weakly stained in cells in which the old macronucleus is located in the center of the cell. New macronuclei are clearly observed in cells in which the old macronucleus has moved to the posterior of the cell (.apprx. 8 h). During later stages of conjugation (10-16 h), the intensity of hv1 staining in new macronuclei increases with time corresponding to the increasing DNA content of these nuclei. Disappearance of detectable hv1 from old macronuclei begins nearly 1 h after these nuclei reach the posterior cytoplasm (.apprx. 9-10 h) and is sometimes complete before these nuclei are eliminated from the cells. Autoradiography of cells labeled for brief periods with [3H]uridine shows that new macronuclei begin to synthesize RNA very soon after the 2nd postzygotic division (.apprx. 8 h). During stages when hv1 is clearly detected in new macronuclei, anlagen are active in RNA synthesis. RNA synthesis in old macronuclei ceases very close to the time when RNA synthesis begins in new macronuclei. The addition of hv1 coincides closely with the transformation of a transcriptionally inactive germinal nucleus into that of a transcriptionally active somatic nucleus. Addition of hv1 may play a fundamental role in macronuclear differentiation and the ability of these cells to carry out transcriptional activity.