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PubMedDate not supplied

Evolution of amitosis of the ciliate macronucleus: gain of the capacity to divide.

Ciliates exhibit nuclear dimorphism, i.e. they have a germline micronucleus and a somatic macronucleus. Macronuclei are differentiated from mitotic sisters of micronuclei. The macronuclei of "higher ciliates" are polyploid and divide acentromerically ("amitotically"); they differentiate once per life cycle. By contrast, Karyorelict (KR) ciliate macronuclei are nearly diploid and cannot divide; they must differentiate at every cell cycle. Diverse lines of evidence are presented to support the hypothesis that ancestral ciliate macronuclei were incapable of division (as in living karyorelict ciliates) and that higher ciliates gained, perhaps independently more than once, the ability to divide the macronucleus. Selective pressures that could have driven the evolution and macronuclear division and two plausible step-wise pathways for the evolution of macronuclear division are proposed. These hypotheses are relevant to our understanding of amitosis mechanisms, evolution of nuclear dimorphism, and phylogenetic classification of ciliates.

Animals↗
PubMedDate not supplied

Conjugation-specific genes in the ciliate Euplotes crassus: gene expression from the old macronucleus.

Following mating or conjugation, the hypotrichous ciliate Euplotes crassus undergoes a massive genome reorganization process. While the nature of the rearrangement events has been well studied, little is known concerning proteins that carry out such processes. As a means of identifying such proteins, differential screening of a developmental cDNA library, as well as construction of a cDNA subtraction library, was used to isolate genes expressed only during sexual reproduction. Five different conjugation-specific genes have been identified that are maximally expressed early in conjugation, during the period of micronuclear meiosis, which is just prior to macronuclear development and the DNA rearrangement process. All five genes are retained in the mature macronucleus. Micronuclear, macronuclear, and cDNA clones of one gene (conZA7) have been sequenced, and the results indicate that the gene encodes a putative DNA binding protein. In addition, the presence of an internal eliminated sequence in the micronuclear copy of the conZA7 gene indicates that this conjugation-specific gene is transcribed from the old macronucleus.

Amino Acid Sequence↗
PubMedDate not supplied

Quantitative changes in periplasmic proteins of the macronucleus-specific bacterium Holospora obtusa in the infection process of the ciliate Paramecium caudatum.

The Gram-negative bacterium Holospora obtusa is a macronucleus-specific symbiont of the ciliate Paramecium caudatum. The infectious form of this bacterium infects the host macronucleus through digestive vacuoles and differentiates into the reproductive form two days after the infection in the nucleus. The monoclonal antibodies IF-3-1 and IF-3-2 reacted with 39 and 15 kDa periplasmic proteins, respectively, that were specific for the infectious form of H. obtusa. Because the antigens were not detected in the reproductive form of the bacterium, it appears that expression of the proteins decreases during or soon after the infection. Using these antibodies, quantitative changes in the antigens in the early infection process were examined by immunoblotting and immunogold electron microscopy. Immunoblotting showed that the amounts of both antigens were reduced within 1 h after the bacteria were engulfed into the digestive vacuoles of the paramecia, but that the amounts of IF-3-2 antigens declined earlier than the IF-3-1 antigen. Immunogold labeling showed that the level of IF-3-2 antigens became very low in the bacteria in the host digestive vacuoles, whereas there was no similar decrease in amount of IF-3-1 antigens. Possible functions of the antigens are discussed. The IF-3-1 antigens decrease in concentration in parallel with the decrease in the periplasmic region.

Animals↗
PubMedDate not supplied

Structure and expression of a GroE-homologous operon of a macronucleus-specific symbiont Holospora obtusa of the ciliate Paramecium caudatum.

The reproductive form of a macronucleus-specific symbiont Holospora obtusa, when harbored by the macronucleus of the ciliate Paramecium caudatum, selectively synthesized a 63-kDa protein which is immunologically related to GroEL, or HSP60, of Escherichia coli. Heat shock treatment of isolated cells of the reproductive and infectious form of the bacterium also induced the synthesis of the GroEL homolog. Immunoblotting showed that the amount of this protein per cell, whether the reproductive or infectious form, is roughly constant. Cloning and sequencing of a gene coding for the GroEL homolog suggested that the protein is 55.2% identical to GroEL of E. coli at the amino acid sequence level, and that the gene is preceded by an open reading frame which encodes a protein 39.6% identical to GroES of E. coli. Northern blot hybridization showed that the groEL homologous gene is highly expressed in the reproductive form, but only in a trace amount in the intermediate and infectious form. Immunoelectron microscopy revealed that the GroEL homolog is localized in the cytoplasm of the reproductive and infectious form.

Amino Acid Sequence↗
PubMed2000

Developmentally regulated rpd3p homolog specific to the transcriptionally active macronucleus of vegetative Tetrahymena thermophila.

A clear relationship exists between histone acetylation and transcriptional output, the balance of which is conferred by opposing histone acetyltransferases (HATs) and histone deacetylases (HDACs). To explore the role of HDAC activity in determining the transcriptional competency of chromatin, we have exploited the biological features of Tetrahymena as a model. Each vegetative cell contains two nuclei: a somatic, transcriptionally active macronucleus containing hyperacetylated chromatin and a transcriptionally silent, germ line micronucleus containing hypoacetylated histones. Using a PCR-based strategy, a deacetylase gene (named THD1) encoding a homolog of the yeast HDAC Rpd3p was cloned. Thd1p deacetylates all four core histones in vitro. It resides exclusively in the macronucleus during vegetative growth and is asymmetrically distributed to developing new macronuclei early in their differentiation during the sexual pathway. Together, these data are most consistent with a potential role for Thd1p in transcriptional regulation and suggest that histone deacetylation may be important for the differentiation of micronuclei into macronuclei during development.

Amino Acid Sequence↗
PubMed1989

Microinjection of plasmid DNA encoding the A surface antigen of Paramecium tetraurelia restores the ability to regenerate a wild-type macronucleus.

Strain d48 of Paramecium tetraurelia contains the A i-antigen gene in the micronucleus, but the gene is lost when micronuclear products develop into the macronucleus. It has recently been shown that when injected into d48, macronucleoplasm from the wild type transforms d48 cells to wild type. It is shown here that wild-type cytoplasm can also bring about transformation, with a marked stage-specific sensitivity for both donor and recipient. It was also found that a plasmid containing the cloned A gene could transform d48 to wild type. Injection of nucleoplasm from animals in the vegetative stage of the cell cycle into the cytoplasm of recipients at various stages of autogamy caused high-frequency transformation of cells able to express the A serotype both before and after the next autogamy. Injection of nucleoplasm into vegetative macronuclei produced over 70% transformants able to express the A serotype after the next autogamy. The ability of nucleoplasm to transform was acquired at the second cell cycle after autogamy and was maintained throughout the vegetative stage. When cytoplasm was obtained from donors during autogamy and injected into the cytoplasm of recipients 1 to 2 h after the sensitive period, quite high frequencies of stable revertants were found when tested both before and after the next autogamy. Cells that were injected into the macronucleus with the cloned A plasmid expressed the A serotype after five fissions in over 20% of the lines and maintained this ability through successive fissions; all transformants except one stably expressed the A serotype even after the next autogamy.

Animals↗
PubMed1983

Infection of macronuclear anlagen of Paramecium caudatum with the macronucleus-specific symbiont Holospora obtusa.

The gram-negative bacterium Holospora obtusa is an endonuclear symbiont of Paramecium caudatum, which is incorporated into the host cells via the food vacuoles and infects their macronucleus exclusively, but never the micronucleus. Since these two kinds of nuclei originate from a fertilization nucleus, it is assumed that the macronucleus acquires a property necessary for it to be recognized by the bacterium at a certain time during the nuclear differentiation process. We found that this property is acquired by four of the eight postzygotic nuclei as soon as the four nuclei differentiate morphologically into the macronuclear anlagen.

Animals↗
PubMed1999

Novel aberrant nuclear behavior in abortive conjugation of Tetrahymena thermophila: joint selection of a meiotic product and macronucleus during nuclear selection.

In conjugation of Tetrahymena thermophila, the paroral zone, cortical cytoplasm in the vicinity of the cytostome, is the site where nuclear selection occurs; one of the four meiotic products is selected in this site prior to the production of gametic pronuclei. During inbreeding cross experiments, several sterile strains were obtained which showed aberrant nuclear behavior. Conjugants of these strains normally underwent meiosis, resulting in the generation of four meiotic products. They, however, failed to complete the process of nuclear selection and aborted the subsequent conjugation sequences. During nuclear selection, macronucleus was frequently selected instead of a meiotic product. A novel aberrant nuclear behavior was observed: Macronucleus and a meiotic product were jointly selected and the both nuclei simultaneously attached to the same paroral zone. When this simultaneous attachment occurred in one partner cell of a pair, nuclear selection was never observed in the other partner cell. This result suggests that a conjugating pair has only two attachment sites on the paroral zone during nuclear selection, and that the distribution of the sites is occasionally distorted in abortive conjugation.

Animals↗
PubMed1992

Bacteriophage lambda DNA fragments replicate in the Paramecium macronucleus: absence of active copy number control.

We show that bacteriophage lambda DNA fragments microinjected into the macronucleus of the ciliated protozoan Paramecium can replicate as unit-length linear molecules. These linear DNA molecules are substrates for the addition of Paramecium telomeres by an endogenous telomerase. The linear DNA pieces can exist at copy numbers much higher than that of typical endogenous macronuclear chromosomes. We show that the copy number of injected DNA many fissions after microinjection reflects that of the original input copy number, suggesting that active control of copy number does not occur. Instead, the results suggest that injected DNA is replicated once per cell division.

Animals↗
PubMed1991

A cdc2-like kinase phosphorylates histone H1 in the amitotic macronucleus of Tetrahymena.

Genetic and biochemical studies have shown that cdc2 protein kinase plays a pivotal role in a highly conserved mechanism controlling the entry of cells into mitosis. It is generally believed that one function of cdc2 kinase is to phosphorylate histone H1 which in turn promotes mitotic chromosome condensation. However, direct evidence linking H1 phosphorylation to mitotic chromatin condensation is limited and the exact cellular function(s) of H1 phosphorylation remains unclear. In this study, we show that mammalian cdc2 kinase phosphorylates H1 from the amitotic macronucleus of Tetrahymena with remarkable fidelity. Furthermore, we demonstrate that macronuclei from Tetrahymena contain a growth-associated H1 kinase activity which closely resembles cdc2 kinase from other eukaryotes. Using polyclonal antibodies raised against yeast p34cdc2, we have detected a 36 kd immunoactive polypeptide in macronuclei which binds to Suc1 (p13)-coated beads and closely follows H1 kinase activity. Since macronuclei divide without mitotic chromosome condensation, these data demonstrate that H1 phosphorylation by cdc2 kinase may be necessary, but is not sufficient to promote mitotic chromatin condensation. The fact that an activity which strongly resembles mammalian cdc2 kinase is active during cell growth in a nucleus which does not undergo mitosis and chromosome condensation suggests that other factors are needed for a true mitotic division to occur. These data also reinforce the notion that H1 phosphorylation has important functions outside mitosis both in Tetrahymena and in mammalian cells.

Animals↗
PubMed1977

The replication of ribosomal DNA in the macronucleus of Tetrahymena.

The replication of extrachromosomal rDNA molecules from the macronucleus of Tetrahymena was studied by electron microscopy. Replication begins in the center of the palindromic molecule and proceeds by means of bidirectional fork movement toward the free ends of the molecule.

Bromodeoxyuridine↗
PubMed1980

Chromatin elimination and the genetic organisation of the macronucleus in Tetrahymena thermophila.

In exponentially growing Tretrahymena thermophila the DNA content of the following structures was determined by cytophotometry: macronuclei of sister cells immediately after division; micronuclei; extranuclear chromatin in dividing cells and postdividers. Further, the development of macronuclear DNA amount in successive cell generations was determined. It was found that chromatin elimination is a frequent process reducing DNA content by about 4% per fission. This chromatin disappears within 20 min after division. The quantity of DNA extruded is highly variable and is different from the micronuclear DNA amount of multiples of it. The frequency of generations with two replication rounds as well as those without replication is estimated to be in the range of 2% each. These findings together with the qualitative difference between micro- and macronuclear DNAs suggest that the macronucleus of Tetrahymena is not entirely composed of complete genomes and that parts of the genetic material must be treated specifically for different sequences either during extrusion or during replication.

Animals↗
PubMed1996

Timing of differential amplification of macronucleus-destined sequences during macronuclear development in the hypotrichous ciliate Euplotes crassus.

The change in copy numbers of macronucleus-destined gene sequences was followed in anlagen DNA during postconjugational development in Euplotes crassus. As noted earlier, copy numbers increase during the polytene stage. During this replication process major differential amplification of different genes is not observed. Instead it is only achieved during or shortly after the fragmentation of the polytene chromosomes. This process is not totally synchronous with respect to different genes. Highly amplified genes are excised earlier than genes with a low final macronuclear copy number. Unexpectedly, the pattern of processing of the newly added oversized telomeres also appears to correlate with the degree of gene amplification. These observations are discussed in terms of a limited replication period after polytene chromosome fragmentation leading to preferential amplification of early excised genes.

Animals↗
PubMed1975

A very rapidly migrating f1 histone associated with gene-sized pieces of DNA in the macronucleus of Oxytricha sp.

Histones bound to gene-sized pieces of DNA in the macronucleus of Oxytricha sp. consist of five main fractions: f1, f2a1, f2a2, f2b and f3. Although the slightly lysine-rich and the arginine-rich histones in Oxytricha macronuclei are similar to comparable fractions in vertebrates, the lysine-rich f1 fraction differs. Oxytricha f1 is unique among eukaryotes in that it migrates faster than f2a1 and is located nearest the cathode in polyacrylamide-urea gels.

Amino Acids↗
PubMed1994

The actin II-encoding gene in the macronucleus of Oxytricha nova.

The macronucleus of the hypotrichous ciliate Oxytricha nova contains two DNA molecules with sizes of approx. 1.4 (actin II) and approx. 1.6 (actin I) kb that encode actin (Act) proteins. The 1.6-kb molecule has been previously described [Greslin et al., DNA 7 (1988) 529-536]. The 1.4-kb molecule is described here. The cloned molecule is 1393 bp in length, including 20 bp of telomere sequence at each end. It has an open reading frame (ORF) of 1119 bp that encodes a putative polypeptide of 374 amino acids (aa) designated as ActII. No introns are present. The coding segment is preceded at its 5' end by an A + T-rich 5' noncoding segment of 100 bp, excluding the telomere, and is followed by an A + T-rich 3' noncoding segment of 124 bp, also excluding the telomere. The aa sequence of the actII gene is 63% identical to actI of O. nova and 68, 70 and 69% identical to the rabbit, yeast and Tetrahymena Act aa sequences, respectively. ActI in O. nova is 64 to 68% identical to Act in other eukaryotes. Thus, the two Act in O. nova differ substantially from one another and neither conforms to the conserved Act sequence generally present in eukaryotes.

Actins↗
PubMed1994

A gene-sized DNA molecule encoding the catalytic subunit of DNA polymerase alpha in the macronucleus of Oxytricha nova.

We have isolated a gene-sized molecule encoding the catalytic subunit of DNA polymerase alpha from a macronuclear genomic library of Oxytricha nova, by using a 0.7-kb fragment of the corresponding human gene as a hybridization probe. Two different versions of the gene are present in the macronucleus, one with an EcoRI site (RI+) and one without an EcoRI site (RI-). The cloned RI- version has been characterized. It is 4938 bp in length, excluding telomeres. It consists of a 329-bp 5' leader, a 4479-bp coding region and a 130-bp 3' trailer. The deduced amino-acid sequence shares conserved regions with the yeast and human polypeptides. We also demonstrate by Southern analysis that gene-sized molecules of similar size, homologous to the isolated O. nova gene are present in the mac genome of closely and distantly related hypotrichs.

Animals↗
PubMed2026

Detection of endocytobionts inhabiting the macronucleus of Frontonia paramagna (Ciliophora, Peniculida).

Bacterial endosymbionts of Frontonia, a widely distributed ciliate genus, remain poorly characterized. Here, we investigated the endosymbiotic microbiota of a Shanghai population of Frontonia paramagna using an integrated morphological and molecular approach. Fluorescence in situ hybridization (FISH) targeting the 16S rRNA gene, coupled with V3-V4 high-throughput sequencing, consistently identified Caedimonas as the bacterial symbiont, specifically localized within the host macronucleus. FISH and transmission electron microscopy confirmed this intramacronuclear colonization with high prevalence and revealed that the symbionts lack flagella and R-bodies. Phylogenetic analysis of full-length 16S rRNA gene sequences placed the F. paramagna symbionts within a well-supported clade containing Caedimonas from divergent hosts. Comparative analysis of the 16S rRNA internal excised element (IEE) showed substantial sequence and secondary structural divergence between the Frontonia-associated lineage and other Caedimonas strains from different ciliates. We conservatively designate this lineage as Caedimonas varicaedens Fpa. These distinct molecular features suggest that the diversity and host distribution of Caedimonas are far from fully described, and genomic approaches will be necessary to evaluate species delimitation within the genus and the possible presence, distribution, and horizontal transfer of R-body genetic determinants.

16S rRNA gene↗