No abstract

Histone fractions have been isolated from the macronucleus of Tetrahymena pyriformis. Five classes of macronuclear histone were purified, using a combination of gel exclusion and ion-exchange chromatography, and were examined with respect to their solubility, electrophoretic, chromatographic, and chemical properties. Tetrahymena H4 is very similar to vertebrate H4, except that it exhibits a larger number of acetylated subfractions. In contrast, the other Tetrahymena histones vary more extensively from their calf thymus counterparts. Tetrahymena H3 resembles calf thymus H3 in its solubility properties and is the only macronuclear histone containing cysteine. However, it differs from vertebrate H3 in composition and has a faster electrophoretic mobility on both urea-acrylamide and sodium dodecyl sulfate-acrylamide gel electrophoresis. Tetrahymena H3 also displays a level of acetylation higher than that reported for its vertebrate homologue. Approximately 45% of macronuclear H2B, which resembles calf thymus H2B in composition and solubility, is present in a (mono)acetylated form, not detected in vertebrate somatic H2B. H1, though similar to its calf thymus homologue in solubility, modification (by phosphorylation), and other properties, differs considerably in its content of basic, acidic, and hydrophobic amino acids. Tetrahymena does not contain a histone strictly homologous to H2A. Although macronuclear histone X resembles H2A in chromatographic and some solubility properties more like H2B than H2A. Fraction X is polymorphic in sodium dodecyl sulfate-acrylamide gels, migrating as two distinct molecular forms. While it is possible that one form is H2A-like and the other more H2B-like, the observation that both forms of X behave identically in solubility fractionation schemes makes this unlikely. Fraction X is both phosphorylated and acetylated which, in addition to two molecular forms, results in a characteristic heterogeneous pattern on urea-acrylamide gels. Characterization of the histone complement of this lower eucaryote should contribute to the understanding of the evolution and biological role of these basic proteins. Moreover, this description represents the most extensive analysis to date of the histones associated with an amitotic, genetically active nucleus. It will serve as a reference to which the histones of the morphologically distinct, mitotically dividing, and genetically inactive micronucleus of this organism can be compared.

Histone fraction F2A1 has been isolated and purified from macronuclei of the ciliate Tetrahymena pyriformis . It migrates as a single species on sodium dodecyl sulphate-acrylamide gel electrophoresis, with a molecular weight indistinguishable from that of calf thymus F2A1 . The solubility properties of Tetrahymena F2AI are also similar to those of calf thymus F2A1 . Electrophoretic analyses on urea-acrylamide gels indicate that Tetrahymena F2A1 consists of four or five subspecies, the two fastest having electrophoretic mobilities identical with those of the two major electrophoretically separable forms of calf thymus F2A1 . High resolution (long gel) electrophoresis coupled with incorporation of radioactive acetate both in vivo and in vitro suggest that, as in the case of calf thymus F2A1, differentical acetylation of a parent molecule can explain the observed electrophoretic heterogeneity of Tetrahymena F2A1 . Electrophoretic analysis of histones isolated from the micronucleus, which is genetically less active than the macronucleus, indicates that it contains largely the relatively unacetylated (parent) form of histone F2A1 .

Antibodies directed against whole histone and purified lysine-rich histone H1extracted from 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 macro-and micronuclei were visibly fluorescent in cells stained with anti-whole histone conjugate. However, the anti-H1 conjugate only labeled macronuclei. This in situ demonstration 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 macro-and 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).

Histones were extracted from isolated mouse liver nuclei, and from mouse liver condensed and extended chromatin . Mouse liver histones were found to be very similar to those of calf thymus in their solubility properties, relative electrophoretic mobilities, and molecular weights as determined on SDS-polyacrylamide gels . Quantitative analysis by high-resolution gel electrophoresis demonstrated a remarkable similarity between the histones of condensed chromatin and those of extended chromatin . However, minor differences were found . A unique subspecies was found only in condensed chromatin histone and the relative amounts of fractions F2AI and F2A2 differed in the two types of chromatin . The ratio of the parental to the acetylated form of F2A1 was identical in the two chromatin samples . Since DNA extracted from the condensed chromatin fraction consisted of approximately 50% satellite DNA, the general similarities between the histones of condensed and extended chromatin make it likely that even this simple, highly repetitive DNA is complexed with a number of histone subfractions .