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C D Allis

Publications and source records attributed to C D Allis.

At least 163 records · Page 9Linked to original sources
PubMed1985

Modulation of linker histones during development in Tetrahymena: selective elimination of linker histone during the differentiation of new macronuclei.

Macronuclei of Tetrahymena thermophila contain a typical H1 which has been shown to be missing from micronuclei. Instead, micronuclei contain three unique polypeptides, alpha, beta, and gamma, which are associated with linker regions of micronuclear chromatin. In this report polyclonal antibodies raised against macronuclear H1 are shown to react with alpha, beta, and gamma by immunoblotting analyses. This result suggests that these polypeptides share some common structural feature(s). Also consistent with this result is the finding that both macro- and micronuclei in growing and mating cells stain positively with H1 antibodies by in situ indirect immunofluorescence. However, these analyses demonstrate that the level of linker histone is greatly reduced in the micronucleus of starved cells and in young macronuclear anlagen. These results are in agreement with earlier biochemical studies and together provide strong evidence that dramatic changes in linker histone accompany nuclear differentiation (and dedifferentiation) in Tetrahymena.

Animals↗
PubMed1985

Deposition-related histone acetylation in micronuclei of conjugating Tetrahymena.

Macro- and micronuclei of the ciliated protozoan, Tetrahymena thermophila, afford a unique opportunity to study histone acetylation under conditions where acetylation associated with the regulation of transcription and acetylation associated with the deposition of histones on the DNA are separable. In this study we demonstrate that histone H3 and histone H4 synthesized in young (5 hr) conjugating Tetrahymena are deposited into micronuclei in acetylated forms. Most of the newly synthesized histone H3 migrates as a monoacetylated form while essentially all of the new histone H4 is deposited as a diacetylated species. Since micronuclei replicate rapidly during this stage of the life cycle, but are transcriptionally inactive, these data suggest that histone acetylation is related functionally to histone deposition and chromatin assembly. Pulse-chase experiments show that micronuclei also contain a butyrate-sensitive deacetylase activity(ies) which operates to remove the deposition-related acetate groups from newly synthesized and deposited H3 and H4. This enzymatic activity probably contributes to the steady state level of micronuclear histone acetylation that is low or nonexistent. Thus, evidence is emerging for at least two independent systems of histone acetylation in Tetrahymena. The first system is specific to macronuclei and may be related to gene expression. The second system is common to macro- or micronuclear histones (H3 and H4) and may be related to histone deposition during DNA replication.

Acetylation↗
PubMed1985

RNA and protein synthesis during meiotic prophase in Tetrahymena thermophila.

Tetrahymena is one of the few organisms from which large amounts of precisely staged meiotic material can be obtained. We took advantage of this fact to monitor RNA and protein synthesis during meiosis. The rate of total protein synthesis as well as the synthesis of the majority of heavily labeled conjugation-specific polypeptides (monitored by high resolution two-dimensional gel electrophoresis) was maximal during meiotic prophase. We therefore cloned cDNAs corresponding to genes active during this time. The mRNA levels of three conjugation-specific genes (pC1, pC2, and pC7) and one conjugation-induced gene (pC3) were followed by using the corresponding labeled cDNAs to probe RNA isolated from different times during mating that was also followed cytologically. Synthesis of the conjugation-specific mRNAs was maximal just prior to maximum crescent stage (pachytene). Evidence is presented for transcription by the normally inactive micronucleus just prior to the maximum crescent stage, confirming an earlier report. The significance of these results is discussed.

Animals↗
PubMed1984

Histone rearrangements accompany nuclear differentiation and dedifferentiation in Tetrahymena.

Histone synthesis and deposition into specific classes of nuclei has been investigated in starved and conjugating Tetrahymena. During starvation and early stages of conjugation (between 0 and 5 hr after opposite mating types are mixed), micronuclei selectively lose preexisting micronuclear-specific histones alpha, beta, gamma, and H3F. Of these histones, only alpha appears to accumulate in micronuclear chromatin through active synthesis and deposition during the mating process. Curiously, alpha is not observed (by stain or label) in young macronuclear anlagen (4C, 10 hr of conjugation). Thus, young macronuclear anlagen are missing all of the histones which are known to be specific to micronuclei of vegetative cells. By 14-16 hr of conjugation, we observe active synthesis and deposition of macronuclear-specific histones, hv1, hv2, and H1, into new macronuclear anlagen (8C). Thus macronuclear differentiation seems well underway by this time of conjugation. It is also in this time period (14-16 hr) that we first detect significant amounts of micronuclear-specific H1-like polypeptides beta and gamma in micronuclear extracts. These polypeptides do not seem to be synthesized during this period, which suggests that beta and gamma are derived from a precursor molecule(s). Since these micronuclear-specific histones do not appear in micronuclear chromatin until after other micronuclei have been selected to differentiate as macronuclei, we suspect that micronuclear differentiation is also an important process which occurs in 10-16 hr mating cells. Our results also suggest that proteolytic processing of micronuclear H3S into H3F (which occurs in a cell cycle dependent fashion during vegetative growth) is not operative during most if not all of conjugation. Thus micronuclei of mating cells contain only H3S which also seems consistent with the fact that some micronuclei differentiate into new macronuclei (micronuclear H3S is indistinguishable from macronuclear H3). Interestingly, the only H3 synthesized and deposited into the former macronucleus of mating cells is the relatively minor macronuclear-specific H3-like variant, hv2. These results demonstrate that significant histone rearrangements occur during conjugation in Tetrahymena in a manner consistent with the fact that during conjugation some micronuclei eventually differentiate into new macronuclei. Our results suggest that selective synthesis and deposition of specific histones (and histone variants) plays an important role in the nuclear differentiation process in Tetrahymena.

Animals↗
PubMed1984

Proteolytic processing of micronuclear H3 and histone phosphorylation during conjugation in Tetrahymena thermophila.

During vegetative growth, micronuclei of the ciliated protozoan Tetrahymena thermophila contain two electrophoretically distinct forms of H3, H3S and H3F [4, 5]. Of these two forms, H3F is unique to micronuclear chromatin and is derived from H3S by a physiologically regulated proteolytic processing event [5]. While the function of this processing event is not clear, several lines of evidence [2, 5] suggest that it may be related to chromatin condensation during mitosis. In this report pulse-chase experiments have been used to study the processing of H3S into H3F during the sexual phase of the life cycle, conjugation. Our results demonstrate that even though micronuclei divide mitotically (and meiotically) several times during the mating process, processing of H3S into H3F does not occur. Failure of H3S to be converted into H3F during these divisions causes a significant increase in the amount of H3S (relative to H3F) as conjugation proceeds. By 10 h of conjugation, essentially all of the micronuclear H3 is in the form of H3S (also see [3]). As long as mating cells are maintained under starvation conditions, processing of H3S into H3F does not occur. However, if exconjugants are returned to food and allowed to proceed through the first true cell division following exconjugation, processing of H3S into H3F occurs. Thus, the return of the processing of H3(3) into H3F following conjugation seems to be tightly coupled to a division which is part of a cell division cycle (as appears to be the case with vegetatively growing cells). The relevancy of these results to the differentiation of new macro- and micronuclei is discussed. H3F is specifically phosphorylated in growing cells, and it has been suggested that this phosphorylation event may be related to chromatin condensation during mitosis [2]. Since in mating cells H3S becomes the more predominant form of H3, the pattern of histone phosphorylation was examined during stages of conjugation where micronuclei are active in mitotic division (6-7 h). While a low level of phosphate label is observed over H3S in mating cells, more phosphate label is associated with the small amount of H3F which remains in micronuclei at this stage of conjugation. We also observe significant amounts of phosphate label associated with micronuclear H2A, H2B, and H4 and each of the micronuclear H1-like molecules, alpha, beta and gamma.

Animals↗
PubMed1984

Specific DNA rearrangements in synchronously developing nuclei of Tetrahymena.

Specific rearrangement of internal chromosomal regions occurs during development of the somatic macronucleus in Tetrahymena thermophila and results in elimination of germ-line (micronuclear) DNA sequences. The timing and mechanism of genome rearrangement within one particular 9.3-kilobase region, which contains three distinct eliminated sequences, were investigated. Portions of this cloned region were used as probes in Southern hybridization experiments to analyze DNA from developing macronuclei (anlagen). All three deletions were found to occur predominantly within a 2-hr time period in which the nuclear DNA contents increased from 4C to 8C (1C represents the amount of DNA present in a haploid genome). The three deletion events can occur independently because intermediate forms, having sustained one or two deletions, were detected. One of the deletions occurs in two alternative ways, resulting in two equally abundant products of different size. Because reciprocal products expected from unequal sister chromatid exchange were not detected, an intramolecular DNA splicing mechanism is suggested.

Animals↗
PubMed1984

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

Vegetative cells of the ciliated protozoan Tetrahymena thermophila contain a transcriptionally active macronucleus and a transcriptionally inactive micronucleus. Earlier studies ( Allis , C. D., C. V. C. Glover , J. K. Bowen, and M. A. Gorovsky , 1980, Cell, 20:609-617; and Allis , C. D., Y. S. Ziegler , M. A. Gorovsky , and J. B. Olmsted, 1982, Cell, 31:131-136) demonstrated the existence of a macronuclear-specific histone variant, hv1 , which is enriched in small punctate regions in nucleoli of several mammalian cell lines. These observations suggest that 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. In this study using antiserum specific for hv1 , we 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 hv1 . 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. However, new macronuclei are clearly observed in cells in which the old macronucleus has moved to the posterior of the cell (approximately 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 (approximately 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 second postzygotic division (approximately 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. Thus, the addition of hv1 coincides closely with the transformation of a transcriptionally inactive germinal nucleus into that of a transcriptionally active somatic nucleus. We suspect that addition of hv1 plays a fundamental role in

Animals↗
PubMed1984

Proteolytic processing of h1-like histones in chromatin: a physiologically and developmentally regulated event in Tetrahymena micronuclei.

Micronuclei isolated from growing cells of Tetrahymena thermophila contain three H1-like polypeptides alpha, beta, and gamma. Micronuclei isolated from young conjugating cells (3-7 h) also contain a larger molecular weight polypeptide, X, which is being actively synthesized and deposited into these nuclei (Allis, C. D., and J. C. Wiggins, 1984, Dev. Biol., 101:282-294). Pulse-chase experiments (with growing and conjugating cells) suggested that X is a precursor to alpha and that alpha is further processed to gamma and a previously undescribed and relatively minor species, delta. These precursor-product relationships were supported by cross-reactivity with polyclonal antibodies raised against alpha and peptide mapping. While beta consistently became labeled under chase conditions (both in growing and mating cells), it was not clear whether it is part of the vivo processing event(s) which interrelates X, alpha, gamma, and delta. Beta was not recognized by alpha antibodies. Despite this uncertainty, these results suggest that proteolytic processing serves to generate significant changes in the complement of H1-like histones present in this nucleus.

Animals↗
PubMed1983

Multiple, independently regulated, polyadenylated messages for histone H3 and H4 in Tetrahymena.

Heterologous probes for yeast H4 and H3 histone genes have been used to study the corresponding histone mRNAs in growing and starved Tetrahymena. Histone mRNAs in both physiological states are polyadenylated. Two types of H4 protein and two types of H3 protein have previously identified in Tetrahymena. Two size classes of H4 messages and three classes of H3 messages have been detected by northern analyses. Southern blot analysis indicate that the number of different kinds of H3 and H4 genes is the same or slightly greater than the number of different messages, suggesting that each message is derived from a different gene. Growing cells have -30 times more histone mRNA than starved cells, even though their total mRNA content is only 4 times greater. The relative abundance of different H4 and H3 messages in growing and starved cells is different, demonstrating that the different messages for a particular type of histone are regulated non-coordinately. In starved cells the presence of a single size class of H3 messages correlates with the preferential synthesis of a previously described macronuclear-specific H3 variant. The fraction of histone messages loaded in growing and starved cells is the same as for bulk mRNAs, and the relative concentrations of the multiple messages for H4 and H3 are the same in polysomal and total RNAs of each cell type. These observations suggest that histone synthesis in Tetrahymena is controlled largely at the level of message abundance, and that very little, if any, control occurs at the translational level.

Animals↗
PubMed1982

Regulation of histone acetylation in Tetrahymena macro- and micronuclei.

Histone acetylation in Tetrahymena macro- and micronuclei has been studied utilizing a combination of electrophoretic and autoradiographic techniques. Histones H2A, H2B, H3, and H4 are acetylated to varying extents in the transcriptionally active macronucleus. There are few, if any, acetylated subspecies of these histones in the transcriptionally inert micronucleus, and micronuclei incorporate little radioactive acetate, either in vivo or in isolated nuclei. Butyrate is shown to inhibit histone deacetylation in Tetrahymena, both in vivo and in isolated nuclei. Incorporation of acetate into micronuclei is unaffected by high concentrations of this inhibitor, indicating that the extremely low levels of histone acetylation observed in micronuclei are not due to rapid deacylation but probably result from the absence of histone acetylation. We also present evidence that macronuclear core histones are composed of at least two classes of molecules distinguishable on the basis of their acetate turnover rates and that individual histone species differ in the distribution of their populations between these classes.

Acetates↗
PubMed1982

A conserved histone variant enriched in nucleoli of mammalian cells.

An antibody has been raised to Tetrahymena histone variant hv1 that specifically stains the macronucleus, but not the micronucleus, of Tetrahymena. This antiserum also stains small punctate regions in nucleoli of several mammalian cell lines. These observations suggest that this histone variant has been highly conserved in evolution and may be associated with transcribed sequences.

Animals↗
PubMed1981

Histone phosphorylation in macro- and micronuclei of Tetrahymena thermophila.

The patterns of histone phosphorylation in amitotically dividing, transcriptionally active macronuclei and in mitotically dividing, transcriptionally inert micronuclei of the ciliated protozoan Tetrahymena thermophila have been analyzed. Taken together, the major phosphorylation events in these two nuclei and their dependence on cell growth and/or division are remarkably similar to those in mammalian cells. Phosphorylation of H1-type proteins occurs in both nuclei and is positively correlated with growth and/or division. Phosphorylation of histone H3 is also positively correlated with growth and/or division but occurs only in micronuclei. Phosphorylation of histone H2A is relatively independent of growth state and occurs largely, if not exclusively, in macronuclei. Given the unique partition of nuclear functions between macro- and micronuclei, these results, coupled with previously reported temporal correlations between specific histone phosphorylations and cell cycle events in mammalian cells [Gurley, L. R., Tobey, R. A., Walters, R. A., Hildebrand, C. E., Hohmann, P. G., D'Anna, J. A., Barham, S. S., & Deaven, L. L. (1978a) in Cell Cycle Regulation (Jeter, J. R., Cameron, J. L., Padilla, G. M. & Zimmerman, A. M. Eds.) pp 37-60, Academic Press, New York], allow insights into the functions of histone phosphorylations. Specifically, a nonmitotic function for extensive H1 phosphorylation and a unique mitotic function for H3 phosphorylation are clearly indicated. A new role for H2A phosphorylation in the regulation of transcriptional activity is also proposed.

Animals↗
PubMed1980

Heat shock, deciliation and release from anoxia induce the synthesis of the same set of polypeptides in starved T. pyriformis.

Heat shock, deciliation and release from anoxia result in similar alterations in the pattern of protein synthesis in starved Tetrahymena pyriformis. In each case, synthesis of the same set of (at least) 16 polypeptides is induced, and many of these polypeptides accumulate in stainable amounts within 50 min. The molecular weights of these proteins, which we refer to as stress proteins (sp), are very similar to those of the heat shock polypeptides of Drosophila. Heat shock and deciliation also lead to similar changes in proteins associated with isolated nuclei. One stress-induced polypeptide, designated sp29c, is highly enriched in the nucleus. This protein is undetectable in control cells but is synthesized in response to stress and accumulates in the nucleus in stainable amounts within 50 min. It is not released by staphylococcal nuclease digestion, suggesting that it is not chromatin-associated. Three other stress-induced proteins, sp73 and sp75a and b, are also present in nuclei isolated from stressed cells but, unlike sp29c, are not enriched in this compartment. Another protein, which is present in stainable quantities in the cytoplasm of control cells, appears to be translocated to the nucleus after stress.

Animals↗
PubMed1980

Proteolytic processing of histone H3 in chromatin: a physiologically regulated event in Tetrahymena micronuclei.

Micronuclei of Tetrahymena thermophila contain two electrophoretically distinct forms of histone H3. The slower migrating micronuclear species, H3S, is indistinguishable from the macronuclear H3 by electrophoretic analyses in three gel systems and by partial proteolytic peptide mapping. The faster species, H3F, is unique to micronuclei. Pulse-chase experiments with radioactive amino acids show that H3S is a precursor to H3F. We present evidence that the in vivo processing of H3S into H3F requires cell growth and/or division and may occur regularly each generation at a specific point in the cell cycle. The processing event must occur after H3F is deposited on micronuclear chromatin, since both H3S and H3F can be isolated from sucrose gradient-purified mononucleosomes (Allis, Glover and Gorovsky, 1979). Partial proteolytic peptide mapping coupled with 3H-N-ethylmaleimide labeling suggest that the processing event involves a proteolytic cleavage from the amino terminal end of H3F. Automated sequence analyses of 14C-lysine-labeled macronuclear H3 together with either 3H-lysine-labeled H3S or H3F demonstrated that H3F is derived from H3S by a proteolytic cleavage which removes six residues from the amino terminus. These observations represent the first demonstration of a physiologically regulated proteolytic processing event in histone metabolism.

Amino Acid Sequence↗