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M A Gorovsky

Publications and source records attributed to M A Gorovsky.

At least 109 records · Page 6Linked to original sources
PubMed1978

Histone-histone interactions in a lower eukaryote, Tetrahymena thermophila.

The six pairwise interactions of Tetrahymena macronuclear histones H2A (formerly HX), H2B, H3, and H4 have been studied using the techniques of circular dichroism and continuous variation. Parallel experiments have been performed with calf thymus histones as controls, and the 12 possible interspecies pairs have also been examined. The behavior of three of the ciliate histones, H2A, H2B, and H3 is virtually identical with that of their vertebrate counterparts. Tetrahymena H4 exhibits a pattern of interactions identical with that of calf thymus H4, but displays significant quantitative differences in the strength of those interactions. Although we have not entirely eliminated the possibility that these differences may be artifactual, we suggest that they reflect the unique primary structure of Tetrahymena H4. These results extend the pattern of histone-histone interactions defined for plants, animals, and fungi to include the protists as well and imply the strong evolutionary conservatism of this pattern in spite of the electrophoretic and compositional differences which exist between homologous histones of different organisms. In addition, they demonstrate conclusively that histone HX should be redefined as Tetrahymena H2A.

Animals↗
PubMed1978

Organization of the 5S RNA genes in macro- and micronuclei of Tetrahymena pyriformis.

The organization of the 5S genes in macro- and micronuclei of Tetrahymena pyriformis was studied using restriction endonucleases. After complete digestion of macronuclear DNA with BamH-I or Hpa I, 5S RNA hybridized to a DNA fragment of approximately 280 base pairs (bp). When macronuclear DNA was only partially digested with these enzymes, hybridization with 32P-5S RNA demonstrated an oligomeric series with a spacing of 280 bp. These results indicate that the 5S genes are tandemly repeated in macronuclei and that the repeating unit is 280 bp (or 180,000 daltons). Since 5S RNA is 120 nucleotides, we conclude that the 5S repeat units contain a 120 bp transcribed region and a 160 bp spacer region. When macronuclear DNA was digested with Eco RI, Bgl I, or Eco RI + Bgl I, 5S RNA hybridized to DNA of molecular weight 3--4 X 10(6), suggesting that these enzymes do not cleave within a 5S repeat. These 3--4 X 10(6) dalton fragments define the maximum size of an average cluster of 5S repeated units. Assuming the size of the 5S repeat to be 0.18 X 10(6) daltons, there are about 15--20 5S repeats per average tanden cluster, and since there are 350 5S-genes per haploid genome, there must be approximately 15--20 tandem arrays. Results obtained using micronuclear DNA suggest that organization of the 5S-genes is very similar in macro- and micronuclei. Macronuclear rRNA genes are extrachromosomal palindromic dimers. In contrast, 5S genes in Tetrahymena were found to be integrated within the genomes of both macro- and micronuclei and not linked to the rRNA genes. Moreover, it is unlikely that they are palindromes; rather they appear to be tandemly repeated in "head-to-tail" linkages. Thus the organization of the 5S genes in Tetrahymena is similar to that of higher eukaryotes.

Animals↗
PubMed1976

Purification and characterization of the histones associated with the macronucleus of Tetrahymena.

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.

Amino Acids↗
PubMed1976

Numbers of 5S and tRNA genes in macro- and micronuclei of Tetrahymena pyriformis.

Macronuclei of Tetrahymena pyriformis contain approximately 200 copies of the genes for 25S and 17S ribosomal RNA (rRNA) per haploid genome. Micronuclei, however, contain only a few copies of the rRNA genes per haploid complement. Since macronuclei develop from products of meiosis, fertilization and division of micronuclei, we suggested that the multiple copies of the rRNA genes in macronuclei are generated by amplification of the small number of genes in micronuclei (Yao et al., 1974). This process provides a simple mechanism for maintaining the homogeneity of the repeated rRNA gnes. To test if amplification is a general mechanism operating on all repeated genes in Tetrahymena, we have examined the numbers of 5S RNA and tRNA genes in macro- and micronuclei. 5S RNA was purified by polyacrylamide gel electrophoresis and hybridized to saturation against macro- and micronuclear DNA. Approximately 0.013-0.014% of macronuclear DNA and about 0.009% of micronuclear DNA is complementary to 5S RNA. After correcting for the differences in the DNA sequence complexities between the two nuclei, we calculate that there are 300-350 5S genes per haploid macro- or micronuclear genome. From these data we conclude that there is little or no detectable amplification of the 5S genes in macronuclei relative to micronuclei. Similar studies using tRNA indicate that these genes are also highly repeated in both nuclei; about 800 genes are present per haploid genome. Thus, amplification from a small number of genes can be excluded as the mechanism for generating the repeated copies of the 5S and tRNA genes in Tetrahymena and it is likely that another, as yet unidentified, mechanism operates to maintain the homogeneity of these genes.

Animals↗
PubMed1976

Subunit structure of rDNA-containing chromatin.

Recent studies indicate that chromatin has a repeating subunit structure. In an attempt to relate this organization to chromatin's role in selective gene transcription we have begun to examine the subunit structure of a specific gene. Tetrahymena pyriformis preferentially replicates the genes coding for rRNA (rDNA) during refeeding after prolonged starvation. By prelabeling cultures during exponential growth with [14C]thymidine and pulse-labeling during refeeding with [3H]thymidine, we have been able to differentially label bulk chromatin and rDNA-containing chromatin. Nuclei which contained at least 78% of their 3H label in rDNA were digested with staphylococcal nuclease, and the DNA digestion products analyzed on agarose gels. Both the kinetics of digestion and the digestion products were similar for 14C- and 3H-labeled chromatin. In order to monitor protein exchange, digestions were also performed on partially purified rDNA-containing chromatin or free rDNA in the presence of nuclei. While the chromatin had a digestion pattern like nuclei, the rDNA was afforded no protection from digestion. Our conclusion is that the chromatin containing rDNA (a repeated, extrachromosomal gene in Tetrahymena) exhibits a particulate structure very similar to that of bulk chromatin. This organization does not exist in free rDNA and is not the result of protein exchange during the nuclease digestion.

Animals↗
PubMed1976

Immunofluorescence evidence for the absence of histone H1 in a mitotically dividing, genetically inactive nucleus.

Antibodies directed against whole histone and purified lysine-rich histone H1 extracted 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-H1 conjugate provide further evidence for the absence of H1 in the genetically inactive, mitotically dividing Tetrahymena micronucleus.

Acridines↗
PubMed1975

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

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 histone 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.

Animals↗
PubMed1975

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

Macronuclei of the ciliated protozoan Tetrahymena pyriformis contain at least five classes of histones, including two with properties like those of histones F3 and F1 of higher eukaryotes. Micronuclei isolated under identical conditions contain little or no detectable F3 or F1. Digestion of both macronuclei and micronuclei with staphylococcal nuclease results in DNA fragments of discrete sizes. The electrophoretic mobilities of the larger fragments suggest that they are oligomers of the smallest ones. These results indicate that the periodic subunit structure observed in the chromatin of higher organisms also occurs in protozoans, and that this structure does not depend on the presence of either histone F1 or F3, even in an organism which has the genetic information for synthesizing these proteins.

Cell Nucleus↗
PubMed1975

Electrophoretic analysis of liver and testis histones of the frog Rana pipiens.

Histones were extracted from frog livers and testes and analyzed by electrophoresis on long polyacrylamide gels and on sodium dodecyl sulfate (SDS)-containing polyacrylamide gels. Frog histones were found to be similar to those of calf thymus except that frog histone fraction F2A2 showed a marked dependence on the temperature at which the long gels were run, and frog histone fraction F3 could be separated from frog F2B on SDS-containing gels. Comparisons between frog liver and frog testis histones indicated that the testis contains as its major F1 component a fast migrating species not found in liver. Testis histones also showed less microheterogeneity of fractions F3 and F2A1 than liver histones. These were the only differences observed between liver and testis histones, even when testis histones were prepared from sperm suspensions that were rich in cells in the late stages of spermiogenesis. Thus it seems that, in Rana, the electrophoretic properties of the basic proteins of sperm differ from those of somatic cells only in the nature of histone F1 and in the degree of microheterogeneity of fractions F2A1 and F3.

Animals↗
PubMed1974

A small number of cistrons for ribosomal RNA in the germinal nucleus of a eukaryote, Tetrahymena pyriformis.

The percentage of DNA complementary to 25S and 17S rRNA has been determined for both the macro- and micronucleus of the ciliated protozoan, Tetrahymena pyriformis. Saturation levels obtained by DNA.RNA hybridization indicate that approximately 200 copies of the gene for rRNA per haploid genome were present in macronuclei. The saturation level obtained with DNA extracted from isolated micronuclei was only 5-10% of the level obtained with DNA from macronuclei. After correction for contamination of micronuclear DNA by DNA from macronuclei, only a few copies (possibly only 1) of the gene for rRNA are estimated to be present in micronuclei. Micronuclei are germinal nuclei. Macronuclei serve as somatic nuclei during vegetative growth but are destroyed every sexual generation and are reformed from products of meiosis, fertilization, and division of the micronuclei. Thus, the hybridization data suggest that the gene for rRNA must be amplified during macronuclear formation with each sexual generation. These observations also demonstrate that the multiple copies of a repeated gene in a somatic nucleus of a eukaryote can be generated from a small number of copies of that gene in a germinal nucleus.

Animals↗
PubMed1974

Histone F1 of Tetrahymena macronuclei: unique electrophoretic properties and phosphorylation of F1 in an amitotic nucleus.

Histone fraction F1 has been isolated and purified from macronuclei of the ciliated protozoan, Tetrahymena pyriformis. In many respects, Tetrahymena F1 is similar to that of other organisms. It is the only Tetrahymena histone soluble in 5% perchloric acid or 5% trichloroacetic acid, has a higher molecular weight than any other Tetrahymena histone, is the histone most easily dissociated from Tetrahymena chromatin, and is susceptible to specific proteolytic cleavage. However, unlike F1 in all other organisms, Tetrahymena F1 is not the slowest-migrating histone fraction when analyzed by polyacrylamide gel electrophoresis at low pH. Tetrahymena F1 also exhibits unusual behavior in sodium dodecyl sulfate-containing polyacrylamide gels, migrating faster than calf thymus F1 at pH 10, and slower than calf thymus F1 at pH 7.6. Tetrahymena F1 was found to be highly phosphorylated in rapidly growing cells, suggesting that the relationship between cell replication and F1 phosphorylation previously observed in mammalian cells may extend to all eukaryotes. The observation that extensive F1 phosphorylation occurs in macronuclei, which divide amitotically, argues against a unique role for F1 phosphorylation in the process of chromosome condensation at mitosis.

Amino Acids↗
PubMed1973

( 6 N)methyl adenine in the nuclear DNA of a eucaryote, Tetrahymena pyriformis.

DNA isolated from macronuclei of the ciliate, Tetrahymena pyriformis, has been found to contain [(6)N]methyl adenine (MeAde); this represents the first clear demonstration of significant amounts of MeAde in the DNA of a eucaryote. The amounts of macronuclear MeAde differed slightly between different strains of Tetrahymena, with approximately 0.65-0.80% of the adenine bases being methylated. The MeAde content of macronuclear DNA did not seem to vary in different physiological states. The level of MeAde in DNA isolated from micronuclei, on the other hand, was quite low (at least tenfold lower than in macronuclear DNA).

Adenine↗
PubMed1973

Studies on histone fraction F2A1 in macro- and micronuclei of Tetrahymena pyriformis.

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 F2A1 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.

Acetates↗