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M A Gorovsky

Publications and source records attributed to M A Gorovsky.

At least 91 records · Page 5Linked to original sources
PubMed1983

The histone content of Tetrahymena ribosomal gene-containing chromatin.

The histone composition of the ribosomal gene containing chromatin (rChromatin) of the ciliated protozoan Tetrahymena pyriformis has been investigated using purified nucleolar fractions in which ribosomal DNA constitutes 65-82% of the total DNA. In isolated nucleoli, rChromatin largely retains the periodic activated structure characteristic of rChromatin in the nucleus. For all five major histone classes, the histone to DNA ratios of nucleolar chromatin are similar to those of bulk macronuclear chromatin. These results argue that the differences between activated rChromatin and inactivated chromatin are not due to a deficiency in the number of histones available to form nucleosomes on the rDNA.

Animals↗
PubMed1982

Histone acetylation and the deoxyribonuclease I sensitivity of the Tetrahymena ribosomal gene.

Under appropriate conditions, up to 8.5% of the total acetate can be removed from the histones of isolated Tetrahymena macronuclei by an endogenous histone deacetylase activity. After in vitro deacetylation, the ribosomal genes are still preferentially digested by DNase I. These observations suggested that either the majority of histone-bound acetate is unnecessary to maintain the DNase I sensitive state or ribosomal chromatin (rChromatin) histones remain acetylated under these conditions. The characteristics of histones acetylation were studied in Tetrahymena rChromatin, which can be isolated in a relatively pure form. Histones associated with the presumably active, DNase I sensitive ribosomal genes have a high steady-state level of histone acetylation which, surprisingly, is maintained by very low acetate turnover rates.

Acetates↗
PubMed1982

Regulation of protein synthesis in Tetrahymena: isolation and characterization of polysomes by gel filtration and precipitation at pH 5.3.

The fraction of ribosomes loaded on polysomes is about 95% in logarithmically growing Tetrahymena thermophila, and about 4% in starved cells. Cytoplasmic extracts from cells in these two physiological states were used to develop column chromatographic methods for the purification of polysomes. Bio-Gel A 1.5 m was found to separate total cytoplasmic ribosomes from many soluble proteins, including RNAse, with no detectable change in the polysome size distribution. Polysomes can be separated from monosomes and non-polysomal mRNA by chromatography on Bio-Gel A 15 m without size selection. These methods can easily be adapted to large scale preparations of polysomes, even from cells where a small fraction of the ribosomes is on polysomes. A method is described for reversible precipitation of polysomes and monosomes from dilute solutions at pH 5.3 which greatly facilitates polysome isolation. Hybridization of 3H-labeled polyU to RNA isolated from column fractions has been used to demonstrate that purification of EDTA released polysomal mRNA can be performed using the column chromatography procedures described here. These methods have been employed to demonstrate that most of the cytoplasmic mRNA in log-phase Tetrahymena is loaded onto polysomes while most of the mRNA is starved cells exists in a non-polysomal form.

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

Nuclease sensitivity of chromatin containing active genes: kinetic analyses utilizing continuous elution of digestion products from an ultrafiltration cell.

Methods have been developed to analyze the kinetics of digestion of chromatin by nucleases. Radioactively labeled nuclei were incubated with enzyme in an ultrafiltration apparatus and digestion rates of different chromatin samples were computed employing a least-squares curve fitting technique to fit the data to zero-order and/or first-order kinetic models. These methods allow detailed kinetic analyses on small amounts of chromatin. Two biological systems were studied. 1) Tetrahymena thermophila macronuclei and micronuclei were compared; these nuclei differ in their transcriptional activities. 2) Ribosomal DNA (rDNA) of Tetrahymena pyriformis, approximately 60% of which codes for rRNA, can be preferentially labeled during starvation-refeeding; its digestion kinetics relative to bulk chromatin were studied. DNase I digested 20-40% of the macromolecular DNA about 3 times faster than bulk macronuclear or micronuclear DNA, and 60-80% of ribosomal gene-containing chromatin about 5 times faster than bulk chromatin. Filter hybridization studies of the DNAase I sensitivity of tRNA, 5S RNA, and ribosomal genes yielded similar results. These data are consistent with the observation that transcribed genes are especially sensitive to attach by DNase I and suggest that activated chromatin structure as probed by extensive DNase I digestion is the same in higher and lower eucaryotes for genes transcribed by all three RNA polymerases. Digestion kinetics of micrococcal nuclease were found to depend on the digestion conditions employed. These two biological systems and the methods we have developed should facilitate analyses of the factors responsible for maintaining an active chromatin structure.

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↗
PubMed1981

Heat shock and deciliation induce phosphorylation of histone H1 in T. pyriformis.

Both heat shock and decilliation of Tetrahymena pyriformis lead to an increase in the level of histone H1 phosphorylation. After heat shock, starved or growing cells reach the same maximum level of H1 phosphorylation, although the increase is more easily detected in starved cells because of their relatively low initial level of phosphorylation. In starved cells, stress-induced phosphorylation is rapid, involves a large percentage of the H1, occurs at multiple sites on the H1 molecule and is inhibited by cycloheximide. Stress-induced phosphorylation of H1 in Tetrahymena thus has many properties in common with cell-cycle-dependent H1 phosphorylation although it is not coupled to the cell cycle.

Animals↗
PubMed1980

DNase I sensitivity of ribosomal genes in isolated nucleosome core particles.

The level of chromatin structure at which DNase I recognizes conformational differences between inert and activated genes has been investigated. Bulk and ribosomal DNA's of Tetrahymena pyriformis were differentially labeled in vivo with [14C]- and [3H]-thymidine, respectively, utilizing a defined starvation-refeeding protocol. The 3H-labeled ribosomal genes were shown to be preferentially digested by DNase I in isolated nuclei. Staphylococcal nuclease digested the ribosomal genes more slowly than bulk DNA, probably owing to the higher GC content of rDNA. DNase I and staphylococcal nuclease digestions of purified nucleosomes and of nucleosome core particles isolated from dual-labeled, starved-refed nuclei were indistinguishable from those of intact nuclei. We conclude from these studies that DNase I recognizes an alteration in the internal nucleosome core structure of activated ribosomal genes.

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↗
PubMed1980

Histone variants specific to the transcriptionally active, amitotically dividing macronucleus of the unicellular eucaryote, Tetrahymena thermophila.

Two dimensional gel electrophoresis (triton-acid-urea followed by SDS) has been used to resolve two previously uncharacterized, quantitatively minor histone variants in acid extracts from macronuclei of Tetrahymena thermophila. Utilizing techniques which allow characterization of these variants without purifying them in significant quantities, we identify one protein as a subtype of H3. The other protein is a moderately lysine-rich histone whose tryptic peptide map differs from that of both H2A and H2B. However, its pattern of secondary modifications, its detergent-binding properties and its methionineless nature all suggest that it is more like H2A than any other histone. Both variants are associated with nucleosomes derived from macronuclei. Thus primary sequence variants of the inner histones, presumably indicative of nucleosome heterogeneity, exist in a lower eucaryote, in an amitotic nucleus, and within the nucleus of a clonally propagated organism. Evidence is presented that these newly described minor variants are absent in micronuclei, suggesting that they play an important role in the structural and functional differentiation of macronuclear chromatin.

Acetylation↗
PubMed1979

Cilia regeneration in starved tetrahymena: an inducible system for studying gene expression and organelle biogenesis.

Deciliated starved Tetrahymena recover motility with kinetics similar to those of growing cells and, like growing cells, require RNA and protein synthesis for regeneration. Comparisons of polysome profiles and electrophoretic analyses of newly synthesized proteins indicate, however, that the basal level of protein synthesis in starved cells is markedly lower than that in growing cells. This difference allows demonstration of changes in protein synthesis following deciliation of starved cells which cannot be detected (if they occur at all) in growing cells. Deciliation of starved cells induces a specific and orderly program of protein synthesis. The synthesis of an 80,000 dalton protein (deciliation-induced protein, DIP) begins shortly after deciliation, comprises 15% of the protein synthesized from 20-60 min, and declines around 60 min after deciliation, shortly after most cells have begun to regenerate cilia. The synthesis of a 55,000 dalton protein is also induced during regeneration and has been identified as tubulin using a well characterized antibody made to ciliary tubulin. Tubulin synthesis is undetectable during the first hour after deciliation even though 60-80% of the cells regain mobility and regenerate short but clearly visible cilia. Tubulin synthesis begins 60 min after deciliation and continues for 2 hr. At its peak, tubulin comprises 7-8% of the protein synthesized. The results of actinomycin D addition at different times after deciliation suggest that RNA required for DIP synthesis is synthesized early (0-30 min), while RNA required for tubulin is synthesized later and over a longer period (30-90 min). Thus deciliation of starved cells, an event occurring at the cell periphery, initiates a well defined and reproducible series of events culminating in cilia formation. This system should be useful in elucidating the molecular mechanisms regulating gene expression and organelle biogenesis in Tetrahymena.

Animals↗
PubMed1979

Micronuclei of Tetrahymena contain two types of histone H3.

Evidence is presented that micronuclei of Tetrahymena thermophila contain significant amounts of two types of histone H3. One is indistinguishable from that found in macronuclei and the other is unique to micronuclei. The micronucleus-specific H3 has a slightly faster mobility than the common H3 in three different gel systems (both of these species were artifactually lost during procedures for histone preparation in previous studies). Both micronuclear H3s appear to contain a single cysteine residue and are present in sucrose gradient-purified nucleosomes. Acid extracts from micronuclei also contain three prominent high molecular weight proteins that also were lost during previous procedures. These proteins are present in extracts from oligomers but are not observed in extracts from mononucleosomes, suggesting that they may be associated with linker regions between nucleosomes.

Animals↗
PubMed1979

Amino-acid sequence of Tetrahymena histone H4 differs from that of higher eukaryotes.

A partial amino-acid sequence of Tetrahymena histone H4 has been determined and differs significantly from the sequence of calf or pea H4. The amino terminus of Tetrahymena H4, unlike that of other H4s so far examined, is not acetylated. Of 66 residues determined, one is a single-residue insertion, one a single-residue deletion, and thirteen are amino-acid replacements with respect to the calf thymus H4 sequence. Most of the amino-acid replacements are nonconservative and are distributed nonrandomly, with a strong concentration in the amino-terminal arm. The first four lysines are partially acetylated. All but two of the replacements can be explained by single nucleotide substitutions at the level of the gene. The similarity in sequence of calf and pea H4 coupled with the substantial differences displayed by Tetrahymena H4 suggest that the divergence of protozoa and higher eukaryotes substantially antedates the divergence of plants and animals. Furthermore, quantitative analysis of the data requires either that the rate of H4 evolution be considerably more rapid than previously thought or that the rate be different for different periods of evolution or for different lines of descent.

Acetylation↗