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PubMed2002

ASI1, a gene encoding a novel leucine zipper protein, is induced during development of the macronucleus in Tetrahymena.

Sexual reproduction in the ciliate Tetrahymena follows a complex developmental program involving the sequential regulation of dozens of genes. Genes that are up-regulated during post-zygotic development in Tetrahymena were isolated by subtractive hybridization. Anlagen stage induced gene 1 (ASI1) encodes a 2.8 kb transcript that contains a single intron and is induced during macronuclear development. ASI1 is a single copy gene in both the micronucleus and the macronucleus. It encodes a 95 kDa conceptual protein with a leucine zipper near the amino terminus.

Amino Acid Sequence↗
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↗
PubMed2002

Topological organization of DNA molecules in the macronucleus of hypotrichous ciliated protozoa.

The DNA in the macronucleus of a hypotrichous ciliate occurs as millions of short molecules packed into dense chromatin bodies 0.1-2 microm in diameter. We have studied by electron microscopy the organization of DNA molecules in these chromatin bodies of macronuclei lysed in water at pH 9. Proteinase K treatment of lysed macronuclei progressively releases from chromatin bodies many rosettes of DNA molecules bound at one or both ends to a central core of protein. With longer treatment with proteinase K, rosettes disappear, leaving individual free DNA molecules. We propose that, in the native state, both ends of DNA molecules are bound through telomere-binding protein to a central core to form rosettes. Many rosettes, with collapsed DNA loops, aggregate to form a chromatin body. Chromatin bodies are believed to dissociate into individual collapsed rosettes to form the granules in the forward zone of the replication band. In the rear zone of the band, the rosettes dissociate, presumably as a result of release of telomere-binding protein, which is preliminary to the replication of the DNA molecules.

Animals↗
PubMed1972

Evidence of gene diminution during the formation of the macronucleus in the protozoan, Stylonychia (DNA density-melting curves-micronuclear DNA-polytene chromosomes).

The course of events by which a macronucleus is formed from a micronucleus after conjugation in the ciliated protozoan, Stylonychia, suggests that genetic diminution may occur. This idea is supported by determinations of the density profiles and melting curves for micro- and macronuclear DNAs. Macronuclear DNA consists of a single density component and melts as if it were a single component. Micronuclear DNA consists of four or more density components and melts as if it were a mixture of several DNAs of different base compositions. These data indicate that at least 60% of the micronuclear DNA components are absent from the macronuclear DNA.

Cell Nucleus↗
PubMed1974

Free ribosomal RNA genes in the macronucleus of Tetrahymena.

In the macronucleus of the ciliated protozoan, Tetrahymena pyriformis GL, the genes coding for 17S and 25S rRNA exist as free, extrachromosomal molecules. About 90% of the molecules are linear with a molecular weight of 12.6 x 10(6). Most of the remainder are circles of the same size, or lariats (circles with tails). A few dimers and oligomers are found. The extrachromosomal rDNA of Tetrahymena represents a kind of gene amplification that may be common among primitive eukaryotes.

Animals↗
PubMed1977

Gene-sized DNA molecules of the Oxytricha macronucleus have the same terminal sequence.

The DNA in the macronucleus of the ciliated protozoan Oxytricha exists as small linear molecules with a number average size of about 3000 base pairs. Most, and possibly all, of these DNA molecules contain the same inverted terminal repeat sequence of approximately 26 base pairs. In addition to its terminal location, two inverted copies of this same sequence surround single-strand interruptions within these DNA molecules. This sequence arrangement may function in the processing of these molecules from large chromosomal precursors or in the subsequent replication of these small linear DNAs during cell reproduction.

Base Sequence↗
PubMed1980

A possible skeletal substructure of the macronucleus of Tetrahymena.

Upon removal of chromatin from isolated macronuclei of tetrahymena, residual structures are obtained, the organization of which faithfully reflects the distinctive architecture of the macronucleus. Macronuclei are isolated by a new procedure in which cells are lysed by immersion in citric acid and Triton X-100. This method is rapid and efficient and leaves the nuclear structures stripped of nuclear envelope and nucleoli. The remaining interconnected chromatin bodies are structurally differentiated into a dense outer shell and a fibrillar inner core. The fibrillar component is identified as chromatin because it is removed upon digestion with DNase and extraction with 2 M NaCl. The dense shell of the chromatin body is unaffected by the digestion procedure, which leaves a skeletal structure comprised of hollow spherical bodies. Analysis of the protein composition by SDS acrylamide gel electrophoresis before and after digestion with DNase and RNase and high-salt extraction shows that histones are diminished, whereas the nonhistone protein composition remains unchanged. It was found the DNase not only extracts chromatin but also protects the nonchromatin structure from the otherwise disruptive effects of high-salt extraction. The method used for isolating the nuclei also affects the structure remaining after the digestion procedure the citric acid/Triton X-100 method enhances the stability of the interconnected spherical bodies. The results indicate that the method for isolating nuclei and the procedure by which chromatin is extracted are both major factors contributing to the detection of a possible nonchromatin nuclear skeleton.

Animals↗
PubMed1982

Rearrangement of repeated DNA sequences during development of macronucleus in Tetrahymena thermophila.

Three clones of non-repetitive sequences and six clones containing repetitive sequences were obtained from micronuclear DNA of Tetrahymena thermophila. All the non-repetitive and three repetitive sequences had the same organization in micro- and macronuclear DNAs as revealed by blot hybridization. On the other hand, the remaining three clones with repetitive sequences had apparently different organization in the two nuclear DNAs. All these repetitive sequences showed a smear on the blot in addition to a number of discrete bands when micronuclear DNA was digested with EcoR I. In macronuclear DNAs, these sequences invariably became one or two bands and the smear disappeared. We conclude that, when a macronucleus develops from a micronucleus, the non-repetitive sequences amplify by more than 20 times with relatively few rearrangement, whereas some selected portions of repeated and/or repeat-contiguous sequences are amplified with rather extensive reorganization.

Animals↗
PubMed1983

Methylation of ribosomal RNA genes in the macronucleus of Tetrahymena thermophila.

We have investigated the occurrence of methylated adenine residues in the macronuclear ribosomal RNA genes of Tetrahymena thermophila. It has been shown previously that macronuclear DNA, including the palindromic ribosomal RNA genes (rDNA), of Tetrahymena thermophila contains the modified base N-6-methyladenine, but no 5-methylcytosine. Purified rDNA was digested with restriction enzymes Sau 3AI, MboI and DpnI to map the positions and levels of N-6-methyladenine in the sequence 5' GATC 3'. A specific pattern of doubly methylated GATC sequences was found; hemimethylated sites were not detected. The patterns and levels of methylation of these sites did not change significantly in different physiological states. A molecular form of the rDNA found in the newly developing macronucleus and for several generations following the sexual process, conjugation, contained no detectably methylated GATC sites. However, both the bulk macronuclear DNA and palindromic rDNA from the same macronuclei were methylated. Possible roles for N-6-methyladenine in macronuclear DNA are discussed in light of these findings.

Animals↗
PubMed1991

Telomeres inhibit end to end fusion and enhance maintenance of linear DNA molecules injected into the Paramecium primaurelia macronucleus.

Direct injection into the macronucleus of Paramecium tetraurelia of DNA molecules coding for the A-antigen leads to expression of the gene and autonomous replication. When injected into Paramecium primaurelia DNA from probably any origin, procaryote or eucaryote, can replicate as linear telomerized molecules and the number of copies maintained can be very high (up to 20000 copies). We present here evidence that if the injected linear DNA molecules harbour preexisting telomeres at both extremities they are protected from degradation, the number of DNA molecules maintained being 15- to 30-fold higher than if the molecules are injected without telomeres. Some of the injected molecules replicate as multimers, but, only when the fused ends are devoid of preexisting telomeric repeats.

Animals↗
PubMed1995

Tandem repeats of the 5' non-transcribed spacer of Tetrahymena rDNA function as high copy number autonomous replicons in the macronucleus but do not prevent rRNA gene dosage regulation.

The rRNA genes in the somatic macronucleus of Tetrahymena thermophila are normally on 21 kb linear palindromic molecules (rDNA). We examined the effect on rRNA gene dosage of transforming T.thermophila macronuclei with plasmid constructs containing a pair of tandemly repeated rDNA replication origin regions unlinked to the rRNA gene. A significant proportion of the plasmid sequences were maintained as high copy circular molecules, eventually consisting solely of tandem arrays of origin regions. As reported previously for cells transformed by a construct in which the same tandem rDNA origins were linked to the rRNA gene [Yu, G.-L. and Blackburn, E. H. (1990) Mol. Cell. Biol., 10, 2070-2080], origin sequences recombined to form linear molecules bearing several tandem repeats of the origin region, as well as rRNA genes. The total number of rDNA origin sequences eventually exceeded rRNA gene copies by approximately 20- to 40-fold and the number of circular replicons carrying only rDNA origin sequences exceeded rRNA gene copies by 2- to 3-fold. However, the rRNA gene dosage was unchanged. Hence, simply monitoring the total number of rDNA origin regions is not sufficient to regulate rRNA gene copy number.

Animals↗
PubMed1979

Extrusion of DNA from the macronucleus of Tetrahymena pyriformis GL.

Administration of the thymidine analog 5-bromodeoxyuridine to exponentially growing cultures of Tetrahymena pyriformis GL in chemically defined medium results in inhibition of cell multiplication by at least one generation before DNA synthesis stops. Cell multiplication can be restored in these cultures, if they are transferred to fresh growth medium, but although most of the cells in the culture contain close to a G2-amount of DNA, a full DNA replication round is a prerequisite for renewed cell multiplication. Large extrusion bodies are found at the first division after transfer to fresh growth medium. Autoradiographic analysis has revealed that the DNA in the extrusion body is a representative of the DNA in the macronucleus indicating a random distribution of DNA between daughter nuclei and extrusion body.

Animals↗
PubMedDate not supplied

conZA8 encodes an abundant protein targeted to the developing macronucleus in Euplotes crassus.

During macronuclear development in the ciliate Euplotes crassus, micronuclear-derived chromosomes undergo a series of rearrangements that include polytenization, DNA splicing, chromosome fragmentation, and telomere addition and processing. Although cis-acting signals that may function in the regulation of these events have been characterized, the proteins that mediate these events have not yet been identified. To identify development-specific factors that may be involved in DNA rearrangement, we previously isolated clones of a number of genes that are expressed only during early macronuclear development. Here, we report the genomic and cDNA sequences of one of these genes, conZA8. The analysis indicates that the conZA8 gene encodes a novel, 468-amino acid, proline-rich protein. Antibodies were raised against both a recombinant form of the conZA8 protein and an internal peptide. Immunoblotting and immunofluorescence analyses indicated that the conZA8 protein is highly abundant, expressed only during the polytene chromosome stage of macronuclear development, and localized to the developing macronucleus. Possible functions of the conZA8 protein are discussed.

Amino Acid Sequence↗
PubMed1990

Changes in fine structure and polypeptide pattern during development of Holospora obtusa, a bacterium infecting the macronucleus of Paramecium caudatum.

The development of the bacterium Holospora obtusa, which infects the macronucleus of Paramecium caudatum, was investigated in the course of a new infection from the infectious form into the reproductive form and vice versa. In parallel with a complete structural reorganization of the bacterium, the protein pattern changed gradually in this development. During the differentiation of the infectious form into the reproductive form, the voluminous periplasm was gradually reduced and the cytoplasm expanded, until the entire bacterium was filled by the cytoplasm. At this stage the long cell divided into five to seven short cells and thereby established the reproductive form, the main stage of the bacterium being maintained and multiplying in the host nucleus. In parallel with the reduction of the periplasm, some of the main proteins of the infectious form gradually disappeared in the electrophoresis pattern; some proteins disappeared earlier than others. Simultaneously, other proteins appeared and gradually became more prominent in the pattern of the developing reproductive form. In the reverse development, when the reproductive form differentiated into the infectious form, the bacterium grew longer, the cytoplasm was condensed, and electron-dense material was deposited in the extending periplasmic space. In parallel with this morphological development, the polypeptide pattern reverted to that of the infectious form.

Animals↗
PubMed1995

Replication of an rRNA gene origin plasmid in the Tetrahymena thermophila macronucleus is prevented by transcription through the origin from an RNA polymerase I promoter.

In the somatic macronucleus of the ciliate Tetrahymena thermophila, the palindromic rRNA gene (rDNA) minichromosome is replicated from an origin near the center of the molecule in the 5' nontranscribed spacer. The replication of this rDNA minichromosome is under both cell cycle and copy number control. We addressed the effect on origin function of transcription through this origin region. A construct containing a pair of 1.9-kb tandem direct repeats of the rDNA origin region, containing the origin plus a mutated (+G), but not a wild type, rRNA promoter, is initially maintained in macronuclei as an episome. Late, linear and circular replicons with long arrays of tandem repeats accumulate (W.-J. Pan and E. H. Blackburn, Nucleic Acids Res, in press). We present direct evidence that the +G mutation inactivates this rRNA promoter. It lacks the footprint seen on the wild-type promoter and produces no detectable in vivo transcript. Independent evidence that the failure to maintain wild-type 1.9-kb repeats was caused by transcription through the origin came from placing a short DNA segment containing the rRNA gene transcriptional termination region immediately downstream of the wild-type rRNA promoter. Insertion of this terminator sequence in the correct, but not the inverted, orientation restored plasmid maintenance. Hence, origin function was restored by inactivating the rRNA promoter through the +G mutation or causing termination before transcripts from a wild-type promoter reached the origin region. We propose that transcription by RNA polymerase I through the rDNA origin inhibits replication by preventing replication factors from assembling at the origin.

Animals↗
PubMed1987

The application of propidium iodide staining to the study of the macronucleus and micronuclei in the suctorian, Heliophrya sp.

Morphological changes in the macronucleus and micronuclei of the ciliated protozoon Heliophrya chapmani were investigated using the nucleic acid-specific stain propidium iodide. The fluorescence patterns of nuclei observed in propidium iodide preparations correspond well with those observed using more conventional DNA-specific methods, such as the Feulgen stain. The advantages of propidium iodide staining (minimal cell loss during staining, rapidity of the staining process, and the avoidance of cell damage during hydrolysis) make this method a quick and efficient alternative in the cytochemical study of the protozoan nucleus.

Animals↗
PubMed1979

[Replicon size and rate of DNA replication in the macronucleus of Tetrahymena pyriformis].

The size of replication units (or replicons) measured in Tetrahymena pyriformis GL macronuclear DNA reaches 20--30 microns, according to the two independent methods: DNA fiber autoradiography, and alkaline isokinetic sucrose gradient centrifugation. The synthesis of new DNA fragments--replicons and their subsequent assembly are separated by time intervals (30 min). The rate of DNA synthesis for one fork averaged 0.6--0.7 microns/min. These data were obtained for cells of cultures being both in the expotential phase of growth, and those synchronized by starvation-refeeding. The generation time of T. pyriformis cells, calculated by the increase of the part of labeled nuclei, is almost 2 hours; the synthesis lasts 1 hour. Total amount of replication units in polyploid (polygenomic) Tetrahymena macronucleus is about 3000. Their initiation during S-period is presumably asynchronous.

Animals↗
PubMed1988

Replication bands and nucleoli in the macronucleus of Euplotes eurystomus: an ultrastructural and cytochemical study.

The principal structural compartments of the macronucleus of Euplotes eurystomus were examined by ultrastructural and cytochemical procedures. Interphase chromatin is condensed in highly compact granules that stain intensely with the DNA-specific osmium-amine procedure. Nucleoli react strongly with silver and with thiol-specific reagents, but are almost completely unstained by osmium-amine. The organelle of DNA synthesis, the replication band, is composed of 2 zones. The forward zone consists of highly ordered chromatin fibers, stains strongly with osmium-amine, with silver, and with thiol-specific reagents. The rear zone, which is the site of DNA synthesis, is impoverished in DNA, and is very sensitive to collapse induced by in vivo heat shock, or during nuclear isolation.

Animals↗