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

1984 ◽  
Vol 98 (6) ◽  
pp. 2107-2117 ◽  
Author(s):  
D Wenkert ◽  
C D Allis
Keyword(s):  
Rna Synthesis ◽  
Tetrahymena Thermophila ◽  
Histone Variant ◽  
Mating Types ◽  
Ciliated Protozoan ◽  
Somatic Nucleus ◽  
Mammalian Cell Lines ◽  
And Function ◽  
Transcribed Sequences ◽  
In Cells

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

Induction of cybrid strains of Tetrahymena thermophila by electrofusion

1988 ◽  
Vol 89 (2) ◽  
pp. 253-261
Author(s):  
J. Gaertig ◽  
M. Kiersnowska ◽  
F. Iftode
Keyword(s):  
Tetrahymena Thermophila ◽  
Mating Types ◽  
Drug Resistant ◽  
Ciliated Protozoan ◽  
Chloramphenicol Resistance ◽  
Electric Pulses ◽  
Conductive Medium ◽  
Membrane Contact ◽  
Initial Process ◽  
Partial Integration

In this paper, the electrofusion of the ciliated protozoan, Tetrahymena thermophila, is described. Deciliated cells were brought into close membrane contact by dielectrophoresis in a weakly conductive medium. Then cell fusion was induced by application of repeated electric pulses. Up to 20 prestarved, logarithmic or stationary phase cells of the same or different mating types may form a single giant cell. The polykaryons are fully able to regenerate cilia and became motile. After an initial process of partial integration the polykaryons yield viable clones by separation of components. Cytoplasmic exchange between fused components occurs before separation. Cytoplasmically inherited chloramphenicol resistance was transmitted from one strain to another by electrofusion and drug-resistant cybrid strains were generated.

scholarly journals FURTHER EVIDENCE FOR LACK OF GENE EXPRESSION IN THE TETRAHYMENA MICRONUCLEUS

Genetics ◽  
1981 ◽  
Vol 98 (4) ◽  
pp. 747-762
Author(s):  
Kristen A Mayo ◽  
Eduardo Orias
Keyword(s):  
Gene Expression ◽  
Rna Synthesis ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Ciliated Protozoan ◽  
Quantitative Evidence ◽  
Soluble Cell ◽  
Cell Extracts ◽  
Radiometric Assay ◽  
Galactokinase Activity

ABSTRACT Certain galA mutations in the ciliated protozoan Tetrahymena thermophila confer an almost total loss of galactokinase activity in homozygotes. Heterokaryons have been constructed that are homogeneous for the galA1 mutation in the (45n) macronucleus, but which contain a galA  + (2n) micronucleus. Soluble cell extracts prepared from these heterokaryons have been assayed for galactokinase activity, using a radiometric assay for the conversion of galactose to galactose-1-phosphate (gal-1-P). No galactokinase activity attributable to the micronuclear genes is observed in such heterokaryons. These results, obtained with the galA1 marker, provide the first direct, quantitative evidence for the lack of micronuclear (germ line) gene expression in Tetrahymena during vegetative growth, and substantiate the predictions of previous phenotypic observations on heterokaryons and autoradiographic studies of micronuclear RNA synthesis. The generality of this conclusion will be established in the future when other enzymically assayable mutations become available for similar studies.

Timing of the appearance of ubiquitinated histones in developing new macronuclei of Tetrahymena thermophila

1991 ◽  
Vol 69 (1) ◽  
pp. 66-71 ◽  
Author(s):  
James R. Davie ◽  
Rueyling Lin ◽  
C. David Allis
Keyword(s):  
Life Cycle ◽  
Western Blotting ◽  
Vegetative Growth ◽  
Tetrahymena Thermophila ◽  
Ciliated Protozoan ◽  
Active Chromatin ◽  
Somatic Nucleus ◽  
Vegetative Cells ◽  
Low Levels

Vegetative cells of the ciliated protozoan Tetrahymena thermophila contain a transcriptionally active macronucleus and a transcriptionally inert micronucleus. During vegetative growth, macronuclear histones H2A and H2B and micronuclear H2A are ubiquitinated. Despite differences in function, macro- and micro-nuclei are related. During conjugation (the sexual phase of the life cycle in Tetrahymena), postzygotic division products of micronuclei give rise to new micro- and macro-nuclei. Using an anti-ubiquitin antibody in Western blotting experiments, we determined the levels of ubiquitinated histones in new macro- and micro-nuclei at various times during conjugation. Very soon after the second postzygotic division (approximately 8 h) when new macronuclei begin to synthesize RNA, ubiquitinated H2B and polyubiquitinated H2A are present. At this time micronuclei have only low levels of ubiquitinated H2A. During later stages of conjugation (15 h), the level of polyubiquitinated H2A decreases, while ubiquitinated H2B increases in developing new macronuclei, attaining levels of ubiquitinated H2B approaching that of parental macronuclei. Ubiquitinated histones are not detectable in the 15-h micronuclei. These results show that ubiquitination of H2B coincides with the transformation of an inert germinal nucleus into that of a transcriptionally active somatic nucleus, suggesting that ubiquitinated H2B has a role in maintaining the transcriptionally active chromatin state.Key words: histone ubiquitination, Tetrahymena thermophila, chromatin, transcription.

Effect of Some Metabolic Inhibitors on Vinblastine-Induced Ribosomal-Granular Material Complexes

1971 ◽  
Vol 29 ◽  
pp. 548-549
Author(s):  
Awtar Krishan ◽  
Dora Hsu
Keyword(s):  
Granular Material ◽  
Rna Synthesis ◽  
Culture Medium ◽  
Actinomycin D ◽  
Metabolic Inhibitors ◽  
Protein And Rna Synthesis ◽  
Apparent Reduction ◽  
Plant Alkaloids ◽  
In Cells

Cells exposed to antitumor plant alkaloids, vinblastine and vincristine sulfate have large proteinacious crystals and complexes of ribosomes, helical polyribosomes and electron-dense granular material (ribosomal complexes) in their cytoplasm, Binding of H3-colchicine by the in vivo crystals shows that they contain microtubular proteins. Association of ribosomal complexes with the crystals suggests that these structures may be interrelated.In the present study cultured human leukemic lymphoblasts (CCRF-CEM), were incubated with protein and RNA-synthesis inhibitors, p. fluorophenylalanine, puromycin, cycloheximide or actinomycin-D before the addition of crystal-inducing doses of vinblastine to the culture medium. None of these compounds could completely prevent the formation of the ribosomal complexes or the crystals. However, in cells pre-incubated with puromycin, cycloheximide, or actinomycin-D, a reduction in the number and size of the ribosomal complexes was seen. Large helical polyribosomes were absent in the ribosomal complexes of cells treated with puromycin, while in cells exposed to cycloheximide, there was an apparent reduction in the number of ribosomes associated with the ribosomal complexes (Fig. 2).

scholarly journals Micro-analysis of pure deoxyribonucleic acid-dependent ribonucleic acid polymerase from Escherichia coli. Action of heparin and rifampicin on structure and function

1970 ◽  
Vol 117 (3) ◽  
pp. 623-631 ◽  
Author(s):  
Volker Neuhoff ◽  
Wolf-Bernhard Schill ◽  
Hans Sternbach
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Deoxyribonucleic Acid ◽  
Structure And Function ◽  
Disc Electrophoresis ◽  
Inhibitory Mechanism ◽  
And Function ◽  
Micro Analysis ◽  
Template Complex

By using micro disc electrophoresis and micro-diffusion techniques, the interaction of pure DNA-dependent RNA polymerase (EC 2.7.7.6) from Escherichia coli with the template, the substrates and the inhibitors heparin and rifampicin was investigated. The following findings were obtained: (1) heparin converts the 24S and 18S particles of the polymerase into the 13S form; (2) heparin inhibits RNA synthesis by dissociating the enzyme–template complex; (3) rifampicin does not affect the attachment of heparin to the enzyme; (4) the substrates ATP and UTP are bound by enzyme loaded with rifampicin; (5) rifampicin is bound by an enzyme–template complex to the same extent as by an RNA-synthesizing enzyme–template complex. From this it is concluded that the mechanism of the inhibition of RNA synthesis by rifampicin is radically different from that by heparin. As a working hypothesis to explain the inhibitory mechanism of rifampicin, it is assumed that it becomes very firmly attached to a position close to the synthesizing site and only blocks this when no synthesis is in progress.

scholarly journals DBC1 (Deleted in Breast Cancer 1) modulates the stability and function of the nuclear receptor Rev-erbα

2013 ◽  
Vol 451 (3) ◽  
pp. 453-461 ◽  
Author(s):  
Claudia C. S. Chini ◽  
Carlos Escande ◽  
Veronica Nin ◽  
Eduardo N. Chini
Keyword(s):  
Breast Cancer ◽  
Nuclear Receptor ◽  
Clock Genes ◽  
Mrna Levels ◽  
Protein Levels ◽  
The Stability ◽  
And Function ◽  
Interfering Rna ◽  
In Cells

The nuclear receptor Rev-erbα has been implicated as a major regulator of the circadian clock and integrates circadian rhythm and metabolism. Rev-erbα controls circadian oscillations of several clock genes and Rev-erbα protein degradation is important for maintenance of the circadian oscillations and also for adipocyte differentiation. Elucidating the mechanisms that regulate Rev-erbα stability is essential for our understanding of these processes. In the present paper, we report that the protein DBC1 (Deleted in Breast Cancer 1) is a novel regulator of Rev-erbα. Rev-erbα and DBC1 interact in cells and in vivo, and DBC1 modulates the Rev-erbα repressor function. Depletion of DBC1 by siRNA (small interfering RNA) in cells or in DBC1-KO (knockout) mice produced a marked decrease in Rev-erbα protein levels, but not in mRNA levels. In contrast, DBC1 overexpression significantly enhanced Rev-erbα protein stability by preventing its ubiquitination and degradation. The regulation of Rev-erbα protein levels and function by DBC1 depends on both the N-terminal and C-terminal domains of DBC1. More importantly, in cells depleted of DBC1, there was a dramatic decrease in circadian oscillations of both Rev-erbα and BMAL1. In summary, our data identify DBC1 as an important regulator of the circadian receptor Rev-erbα and proposes that Rev-erbα could be involved in mediating some of the physiological effects of DBC1.

scholarly journals Evidence for rapid structural and functional changes of the melanophore microtubule-organizing center upon pigment movements

1979 ◽  
Vol 83 (3) ◽  
pp. 623-632 ◽  
Author(s):  
M Schliwa ◽  
U Euteneuer ◽  
W Herzog ◽  
K Weber
Keyword(s):  
Complete Removal ◽  
Cell System ◽  
The State ◽  
Microtubule Assembly ◽  
Microtubule Organizing Center ◽  
Functional Changes ◽  
Organizing Center ◽  
Pigment Distribution ◽  
And Function ◽  
In Cells

Melanophores of the angelfish, pterophyllum scalare, have previously been shown to display approximately 2,400 microtubules in cells wih pigment dispersed; these microtubules radiate from a presumptive organizing center, the central apparatus (CA), and their number is reduced to approximately 1,000 in the state with aggregated pigment (M. Schliwa and U. Euteneuer, 1978, J. Supramol. Struct. 8:177-190). In an attempt to elucidate the factors controlling this rapid reorganization of the microtubule apparatus, structure and function of the CA have been investigated under different physiological conditions. As a function of the state of pigment distribution, melanophores differ markedly with respect to CA organization. A complex of dense amorphous aggregates and associated fuzzy material, several micrometers in diameter, surrounds the centrioles in cells with pigment dispersed, and numerous microtubules emanate from this complex in a radial fashion. In the aggregated state, on the other hand, few microtubules are observed in the pericentiolar region, and the amount of fibrous material is greatly reduced. These changes in CA morphology as a function of the state of pigment distribution are associated with a marked difference in its capacity to initiatiate the assembly of microtubules from exogenous pure porcine brain tubulin in lysed cell preparations. After complete removal of preexisting microtubules, cells lysed in the dispersed state into a solution of 1-2 mg/ml pure tubulin have numerous microtubules associated with the CA in radial fashion, while cells lysed in the aggregated state nucleate the assembly of only a few microtubules. We conclude that it is the activity of the CA that basically regulates the expression of microtubules. This regulation is achieved through a variation in the capacity to initiate microtubule assembly. Increase or decrease in the amount of dense material, as readily observed in the cell system studied here, seems to be a morphologic expression of such a physiologic function.

scholarly journals Constitutive expression, not a particular primary sequence, is the important feature of the H3 replacement variant hv2 in Tetrahymena thermophila.

1997 ◽  
Vol 17 (11) ◽  
pp. 6303-6310 ◽  
Author(s):  
L Yu ◽  
M A Gorovsky
Keyword(s):  
Protein Sequence ◽  
Tetrahymena Thermophila ◽  
Histone H3 ◽  
Constitutive Expression ◽  
Histone Variants ◽  
Wild Type ◽  
Ciliated Protozoan ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Constitutive Synthesis

Although quantitatively minor replication-independent (replacement) histone variants have been found in a wide variety of organisms, their functions remain unknown. Like the H3.3 replacement variants of vertebrates, hv2, an H3 variant in the ciliated protozoan Tetrahymena thermophila, is synthesized and deposited in nuclei of nongrowing cells. Although hv2 is clearly an H3.3-like replacement variant by its expression, sequence analysis indicates that it evolved independently of the H3.3 variants of multicellular eukaryotes. This suggested that it is the constitutive synthesis, not the particular protein sequence, of these variants that is important in the function of H3 replacement variants. Here, we demonstrate that the gene (HHT3) encoding hv2 or either gene (HHT1 or HHT2) encoding the abundant major H3 can be completely knocked out in Tetrahymena. Surprisingly, when cells lacking hv2 are starved, a major histone H3 mRNA transcribed by the HHT2 gene, which is synthesized little, if at all, in wild-type nongrowing cells, is easily detectable. Both HHT2 and HHT3 knockout strains show no obvious defect during vegetative growth. In addition, a mutant with the double knockout of HHT1 and HHT3 is viable while the HHT2 HHT3 double-knockout mutant is not. These results argue strongly that cells require a constitutively expressed H3 gene but that the particular sequence being expressed is not critical.

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