Absence of histone F1 in a mitotically dividing, genetically inactive nucleus.

Abstract

Histones were extracted from macro-and micronuclear chromatin of the ciliated protozoan Tetrahymena pyriformis. Conditions that resulted in macronuclear chromatin containing large amounts of histone F1 yielded micronuclear chromatin in which this histone was absent. Evidence is presented indicating that the absence of F1 from micronuclei is not a preparative artifact and that histone F1 is replaced by other tistone fractions. Since micronuclei divide mitotically, while macronuclei divide amitotically, these results suggest that histone F1 and its phosphorylation do not play an indispensable role in the process of mitotic chromosome condensation, in chromosome replication, or in the separation of newly synthesized chromatids. Like most ciliated protozoans, the vegetative cells of Tetrahymena pyriformis contain a macro-and a micronucleus. These two nuclei are formed from the daughter products of a single mitotic division during the sexual process of conjugation. It is likely, therefore, that they contain the same, or closely related, genetic information. In spite of their common origin and the fact that they reside in the same cytoplasmic millieu, macro-and micronuclei differ considerably in their structure and function (see ref. 1 for review). For example, macronuclei divide amitotically, with no sign of mitotic chromosomes; micronuclei divide mitotically (2-7). In addition, the two nuclei differ greatly in their genetic activity. Macronuclei are sites of intense RNA synthesis while micronuclei synthesize little, if any, detectable RNA (8, 9).We have recently reported that the macronucleus of Tetrahymena contains a histone with properties similar to those of histone F1 of higher organisms (10). Macronuclear F1 isolated from rapidly growing cells was found to be highly phosphorylated, while histones isolated from slowly growing cells contained only small amounts of phosphorylated FL. Thus, the relationship between the rate of cell replication and the extent of phosphorylation of histone F1 observed in a variety of other cell types (11)(12)(13)(14)(15)(16) MATERIALS AND METHODS Cell Culture. Tetrahymena pyriformis, strains WH-6 (Syngen 1, mating type I; a micronucleate strain) and GL (an amicronucleate strain), were cultured as described (27).Isolation of Nuclei and Chromatin. Nuclei were isolated as described (27,28). Chromatin was isolated by a modification of the method of Panyim et al. (29). Chromatin deficient in histone fraction F1 was prepared by washing isolated nuclei repeatedly in 0.5 M NaCl. This treatment was found to remove most of histone F1 from macronuclei while extracting little of the other histone fractions.Isolation of Histones. Histones were isolated from whole nuclei or from chromatin by extraction with either 2.4 M urea/0.2 M H2SO4 or with 0.2 M H2SO4 (10, 27).Thermal Denaturation of Isolated Chromatin. Solubilized macro-or micronuclear chromatin was fixed in 0.91% formaldehyde for at least 15 hr and then dialyzed for at least 5 hr against three changes (100 volumes each) of 0.2 mM ...

Subunit structure of a naturally occurring chromatin lacking histones F1 and F3.

“…We have also presented evidence that micronuclei do not contain histones F3 (this paper) or F1 (25). The absence of F3 in micronuclei which divide mitotically, coupled with the presence of this histone in macronuclei (which divide amitotically) makes it unlikely that this histone plays an indispensible role in mitotic chromosome condensation as has been suggested (33,34).…”

“…of F3 due to the formation of disulfide bonds with other proteins. We have previously described mixing experiments (25) which argue strongly against the possibilities that there are artifactual losses of histones from micronuclei during our isolation procedures. Also, micronuclear chromatin does not appear to be histone deficient when examined by a number of physical and chemical criteria (25).…”

“…We have previously described mixing experiments (25) which argue strongly against the possibilities that there are artifactual losses of histones from micronuclei during our isolation procedures. Also, micronuclear chromatin does not appear to be histone deficient when examined by a number of physical and chemical criteria (25). Rather, it appears that histone F1 and F3 (and Y) are not present in micronuclei and that their absence in micronuclear chromatin is compensated by increased amounts of the other Tetrahymena histones (F2A1, F2B, and X).…”

“…Gels were stained in fast green and were scanned in a Gilford spectrophotometer (22 (27). These (25) or acetylated subspecies of F2A1 (20). In Fig.…”

Isopeptidase: a novel eukaryotic enzyme that cleaves isopeptide bonds.

“…Histone phosphorylation has been implicated in chromatin condensation in mitosis (4-9); however, this idea has been questioned recently (10,11). In attempts to seek feasible mechanisms, we found another mitosis-specific event that involves a reversible stoichiometric shift in one ofthe structural chromatin proteins, A24 (12-14), a conjugate of histone H2A and the nonhistone protein ubiquitin (15-17).…”