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PubMed1986

Inhibition of Paramecium caudatum by an Alteromonas luteoviolacea antibiotic.

An antibiotic factor obtained from Alteromonas luteoviolacea was shown to kill Paramecium caudatum. This substance was shown to be destroyed by boiling, sonication, or freezing. It passed through a 0.45-micron-pore-size filter and was not precipitated from a culture supernatant with ethyl alcohol. Its production varied with A. luteoviolacea strains and medium constituents. It was also shown to be distinct from the A. luteoviolacea antibiotic that affects bacterial cells.

Animals↗
PubMed2004

Epigenetic control of chromosome breakage at the 5' end of Paramecium tetraurelia gene A.

Macronuclei and micronuclei of ciliates have related genomes, with macronuclei developing from zygotic micronuclei through programmed DNA rearrangements. While Paramecium tetraurelia wild-type strain 51 and mutant strain d48 have the same micronuclear genome, qualitative differences between their macronuclear genomes have been described, demonstrating that programmed DNA rearrangements could be epigenetically controlled in ciliates. Macronuclear chromosomes end downstream of gene A (A51 Mac ends) and at the 5' end of gene A (Ad48 Mac ends) in strains 51 and d48, respectively. To gain further insight into the process of chromosome end formation, we performed an extensive analysis of locus A rearrangement in strains d48 and 51, in strain d12, which harbors a gene A deletion, and in interstrain cross progeny. We show that (i) allele Ad12 harbors a deletion of >16 kb, (ii) A51 Mac ends distribute over four rather than three DNA regions, (iii) strains d48 and 51 display only quantitative differences (rare Ad48 and A51 Mac ends do form in strains 51 and d48, respectively), (iv) the level of A51 Mac ends is severalfold enhanced in d12- and d48-derived progeny, and (v) this level inversely correlates with the level of Ad48 Mac ends in the d48 parent. Together, these data lead to a model in which the formation of Ad48 Mac ends is epigenetically controlled by a d48 factor(s). We propose that the d48 factor(s) may be derived from RNA molecules transcribed from the Ad48 Mac ends and encompassing the truncated A gene and telomeric repeats.

Animals↗
PubMed2005

Analysis of Paramecium tetraurelia A-51 surface antigen gene mutants reveals positive-feedback mechanisms for maintenance of expression and temperature-induced activation.

In Paramecium tetraurelia, variable surface antigen loci show mutually exclusive expression which is controlled primarily at the transcriptional level. Clonally stable expression of a single antigen has attracted models involving self-regulation by their gene products. However, direct demonstration of self-feedback at the molecular level has been complicated due to the inability to separate the functional gene from its product as well as copy number effects associated with injected extrachromosomal DNA in the polygenomic somatic nucleus. In this study, we exploited several germ line termination and frameshift mutations in the A-51 surface antigen gene to analyze variable surface antigen expression. These mutant alleles have the same copy number as the wild-type allele and therefore eliminate possible copy number effects. The mutant alleles were not transcribed at 27 degrees C, consistent with positive-feedback models for gene expression. However, further analysis showed that high temperatures (34 degrees C) induced transcription of the mutant A genes even in the presence of a different antigen on the cell surface. Thus, transcription was temperature dependent. Unlike wild-type cells, transcription of the mutant A genes at high temperatures was not maintained after temperature shift back to 27 degrees C in homozygous mutant cells. Importantly, transcription of the mutant allele was maintained at 27 degrees C in heterozygous cells with one copy of the wild-type allele. These results indicate that expression of the wild-type gene is required to stabilize its own transcriptional state at 27 degrees C.

Amino Acid Sequence↗
PubMed2005

Nd6p, a novel protein with RCC1-like domains involved in exocytosis in Paramecium tetraurelia.

In Paramecium tetraurelia, the regulated secretory pathway of dense core granules called trichocysts can be altered by mutation and genetically studied. Seventeen nondischarge (ND) genes controlling exocytosis have already been identified by a genetic approach. The site of action of the studied mutations is one of the three compartments, the cytosol, trichocyst, or plasma membrane. The only ND genes cloned to date correspond to mutants affected in the cytosol or in the trichocyst compartment. In this work, we investigated a representative of the third compartment, the plasma membrane, by cloning the ND6 gene. This gene encodes a 1,925-amino-acid protein containing two domains homologous to the regulator of chromosome condensation 1 (RCC1). In parallel, 10 new alleles of the ND6 gene were isolated. Nine of the 12 available mutations mapped in the RCC1-like domains, showing their importance for the Nd6 protein (Nd6p) function. The RCC1 protein is well known for its guanine exchange factor activity towards the small GTPase Ran but also for its involvement in membrane fusion during nuclear envelope assembly. Other proteins with RCC1-like domains are also involved in intracellular membrane fusion, but none has been described yet as involved in exocytosis. The case of Nd6p is thus the first report of such a protein with a documented role in exocytosis.

Alleles↗
PubMed1970

Action of penicillin G on endosymbiote lambda particles of Paramecium aurelia.

The kinetics of loss from the cytoplasm and changes in ultrastructure of symbiont lambda particles after treatment of axenically cultivated lambda-bearing Paramecium aurelia with penicillin G was investigated. Low concentrations (1 to 2 unit/ml) of the antibiotic caused many particles within the cell to become filamentous; high concentrations (2,000 unit/ml) caused lysis of the particles without noticeably affecting the protozoan. The ED(50) value (2 to 3 unit/ml) was within the range of values found to cause lysis of many gram-negative bacteria. Rapidly dividing lambda were more vulnerable to the action of the antibiotic than slowly dividing particles. Nondividing particles were not affected by exposure to the antibiotic. Ultrastructural changes observed in lambda during lysis by penicillin G were consistent with the view that penicillin interferes with the synthesis of a vital component of the cell envelope of the particle, possibly a peptidoglycan similar to that found in the cell walls of bacteria. The deoxyribonucleic acid of lambda was dispersed throughout the particle as electron dense fibers enclosed within electron transparent areas. The cell envelope appeared to consist of at least two morphologically distinguishable layers, an inner layer homologous to the plasma membrane of bacteria and an outer layer homologous to the bacterial cell wall. Lambda may be regarded as a randomly distributed population of bacteria growing and dividing synchronously within the collective cytoplasm of its protozoan host.

Animals↗
PubMed1977

Chromosomal and extrachromosomal deoxyribonucleic acid from four bacterial endosymbionts derived from stock 51 of Paramecium tetraurelia.

Four variant lines of stock 51 kappa (Paramecium tetraurelia) were screened for the presence of covalently closed circular (CCC) deoxyribonucleic acid (DNA). Stock 51m43 kappa, a nonkiller resistant to 51 killing, contained four classes of CCC DNA: 2.9 X 10(7), 9.7 X 10(7), and 11.8 X 10(7) daltons. The buoyant densities of 51m43 kappa chromosomal and CCC DNA were 1.700 and 1.698 g/cm3, respectively. Stock 51m43 pi, a sensitive nonkiller, contained two CCC species: 0.3 X 10(7) and 4.4 X 10(7) daltons. The buoyant densities of both the chromosomal and CCC DNA were 1.694 to 1.695 g/cm3. Three sizes of CCC DNA were found in 51m1 pi: 0.3 X 10(7), 2.3 X 10(7), and 4.5 X 10(7) daltons. The buoyant densities of both the chromosoaml DNA and the CC DNA were 1.694 to 1.695 g/cm3. It is not known whether 51m1 kappa, a sensitive spinner killer, contains CCC DNA. The buoyant density of its chromosomal DNA was 1.703 g/cm3. Of the four variant lines, only 51m43 kappa appears to be a mutant of 51 kappa. The chromosomal and CCC DNAs of 51m43 kappa have the same buoyant densities as those of 51 kappa; in addition 51m43 kappa contain a CCC molecule the same size as that found in 51 kappa (2.8 x 10(7) daltons). The three other lines are probably bacterial species that are distinct from 51 kappa and which, at one time, were co-inhabitants with 51 kappa in stock 51 paramecia.

Animals↗
PubMed1977

Phylogenetic relationships of bacterial endosymbionts of Paramecium aurelia: polynucleotide sequence relationships of 51 kappa and its mutants.

Hydroxyapatite chromatographic procedures were used to investigate the deoxyribonucleic acid (DNA) sequence relationships of kappa of Paramecium tetraurelia stock 51 and the organisms that have been designated as mutants of 51 kappa. Of the "mutants" studied, only 51m43 kappa possessed a high percentage (89%) of DNA sequences homologous to those of 51 kappa. All other "mutant" strains possessed less than 25% polynucleotide sequence homology to 51 kappa DNA. The three strains of pi endosymbionts (51m1 pi, 51m43 pi, and 139 pi) share greater than 75% DNA sequence homology with each other and approximately 50% DNA sequence homology with 138 mu, the mate-killer endosymbiont found in P. octaurelia. Only 23% of the 51 kappa DNA sequences were found to be homologous with those of 51m1 kappa. The data indicate that of the "mutants" studied, only 51m43 kappa could be a mutant of 51 kappa. The pi endosymbionts comprise a closely related group of organisms that are also related to 138 mu but not to any of the kappas tested. The group of organisms designated as kappa appears to be comprised of at least two distinct phylogenetic groups.

Animals↗
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↗
PubMed1991

Macronuclear transformation with specific DNA fragments controls the content of the new macronuclear genome in Paramecium tetraurelia.

A previously isolated mutant cell line called d48 contains a complete copy of the A surface antigen gene in the micronuclear genome, but the gene is not incorporated into the macronucleus. Previous experiments have shown that a cytoplasmic factor made in the wild-type macronucleus can rescue the mutant. Recently, S. Koizumi and S. Kobayashi (Mol. Cell. Biol. 9:4398-4401, 1989) observed that injection of a plasmid containing the A gene into the d48 macronucleus rescued the cell line after autogamy. It is shown here that an 8.8-kb EcoRI fragment containing only a portion of the A gene coding region is sufficient for the rescue of d48. The inability of other A gene fragments to rescue the mutant shows that this effect is dependent upon specific Paramecium DNA sequences. Rescue results in restoration of the wild-type DNA restriction pattern in the macronucleus. These results are consistent with a model in which the macronuclear A locus normally makes an additional gene product that is required for correct processing of the micronuclear copy of the A gene.

Animals↗
PubMed1997

Sequence-specific epigenetic effects of the maternal somatic genome on developmental rearrangements of the zygotic genome in Paramecium primaurelia.

In ciliates, the germ line genome is extensively rearranged during the development of the somatic macronucleus from a mitotic product of the zygotic nucleus. Germ line chromosomes are fragmented in specific regions, and a large number of internal sequence elements are eliminated. It was previously shown that transformation of the vegetative macronucleus of Paramecium primaurelia with a plasmid containing a subtelomeric surface antigen gene can affect the processing of the homologous germ line genomic region during development of a new macronucleus in sexual progeny of transformed clones. The gene and telomere-proximal flanking sequences are deleted from the new macronuclear genome, although the germ line genome remains wild type. Here we show that plasmids containing nonoverlapping segments of the same genomic region are able to induce similar terminal deletions; the locations of deletion end points depend on the particular sequence used. Transformation of the maternal macronucleus with a sequence internal to a macronuclear chromosome also causes the occurrence of internal deletions between short direct repeats composed of alternating thymines and adenines. The epigenetic influence of maternal macronuclear sequences on developmental rearrangements of the zygotic genome thus appears to be both sequence specific and general, suggesting that this trans-nucleus effect is mediated by pairing of homologous sequences.

Animals↗
PubMed1998

Homology-dependent maternal inhibition of developmental excision of internal eliminated sequences in Paramecium tetraurelia.

Thousands of single-copy internal eliminated sequences (IESs) are excised from the germ line genome of ciliates during development of the polygenomic somatic macronucleus, following sexual events. Paramecium IESs are short, noncoding elements that frequently interrupt coding sequences. No absolutely conserved sequence element, other than flanking 5'-TA-3' direct repeats, has been identified among sequenced IESs; the mechanisms of their specific recognition and precise elimination are unknown. Previous work has revealed the existence of an epigenetic control of excision. It was shown that the presence of one IES in the vegetative macronucleus results in a specific inhibition of the excision of the same element during the development of a new macronucleus, in the following sexual generation. We have assessed the generality and sequence specificity of this transnuclear maternal control by studying the effects of macronuclear transformation with 13 different IESs. We show that at least five of them can be maintained in the new macronuclear genome; sequence specificity is complete both between genes and between different IESs in the same gene. In all cases, the degree of excision inhibition correlates with the copy number of the maternal IES, but each IES shows a characteristic inhibition efficiency. Short internal IES-like segments were found to be excised from two of the IESs when excision between normal boundaries was inhibited. Available data suggest that the sequence specificity of these maternal effects is mediated by pairing interactions between homologous nucleic acids.

Animals↗
PubMed1999

Does ribosomal DNA get out of the micronuclear chromosome in Paramecium tetraurelia by means of a rolling circle?

The macronuclear genes coding for rRNA (ribosomal DNA [rDNA]) of Paramecium tetraurelia, stock 51, are arranged in polymers consisting of units made up of a transcribed coding region and a nontranscribed spacer region. The whole macronuclear polymer ends with a portion of the spacer on either end followed by a telomere. Six kinds of macronuclear units, or genes, were mapped. Spacers were different, and transcribed regions were the same. These genes are found in markedly different numbers in the macronucleus. The most common gene shows two regions in the spacer where a sequence is followed by a direct repeat. The next most common gene is similar but shows a deletion plus a number of base pair substitutions. Although most cosmid clones contain only a single kind of gene, many contain more than one. These are thought to be produced by somatic crossing over. The four micronuclear genes that have been isolated consist of a single central transcribed region and portions of the spacer on either end. Sequencing indicates that the two ends of the molecule are partially redundant. While the spacer region at the right end of the macronuclear polymer is derived from the micronuclear spacer on the right, the spacer at the left end of the macronuclear polymer is derived from regions of the micronuclear spacer on both the right and the left. To account for this situation, a rolling-circle model for generation of the macronuclear rDNA from the micronuclear DNA is proposed.

Animals↗
PubMed1999

Expression of mutated Paramecium telomerase RNAs in vivo leads to templating errors that resemble those made by retroviral reverse transcriptase.

Telomeric DNA consists of short, tandemly repeated sequences at the ends of chromosomes. Telomeric DNA in the ciliate Paramecium tetraurelia is synthesized by an error-prone telomerase with an RNA template specific for GGGGTT repeats. We have previously shown that misincorporation of TTP residues at the telomerase RNA templating nucleotide C52 accounts for the 30% GGGTTT repeats randomly distributed in wild-type telomeres. To more completely characterize variable repeat synthesis in P. tetraurelia, telomerase RNA genes mutated at C52 (A, U, and G) were expressed in vivo. De novo telomeric repeats from transformants indicate that the predominant TTP misincorporation error seen in the wild-type telomerase is dependent on the presence of a C residue at template position 52. Paradoxically, the effects of various other telomerase RNA template and alignment region mutations on de novo telomeres include significant changes in fidelity, as well as the synthesis of aberrant, 5-nucleotide telomeric repeats. The occurrence of deletion errors and the altered fidelity of mutated P. tetraurelia telomerase, in conjunction with misincorporation by the wild-type enzyme, suggest that the telomerase RNA template domain may be analogous to homopolymeric mutational hot spots that lead to similar errors by the human immunodeficiency virus proofreading-deficient reverse transcriptase.

Animals↗
PubMed2000

Developmentally regulated excision of a 28-base-pair sequence from the Paramecium genome requires flanking DNA.

The micronuclear DNA of Paramecium tetraurelia is estimated to contain over 50,000 short DNA elements that are precisely removed during the formation of the transcriptionally active macronucleus. Each internal eliminated sequence (IES) is bounded by 5'-TA-3' dinucleotide repeats, a feature common to some classes of DNA transposons. We have developed an in vivo assay to analyze these highly efficient and precise DNA excision events. The microinjection of a cloned IES into mating cells results in accurately spliced products, and the transformed cells maintain the injected DNA as extrachromosomal molecules. A series of deletions flanking one side of a 28-bp IES were constructed and analyzed with the in vivo assay. Whereas 72 bp of DNA flanking the eliminated region is sufficient for excision, lengths of 31 and 18 bp result in reduced excision and removal of all wild-type sequences adjacent to the TA results in complete failure of excision. In contrast, nucleotide mutations within the middle of the 28-bp IES do not prevent excision. The results are consistent with a functional role for perfect inverted repeats flanking the IES.

Animals↗
PubMed2000

Timing of developmentally programmed excision and circularization of Paramecium internal eliminated sequences.

Paramecium internal eliminated sequences (IESs) are short AT-rich DNA elements that are precisely eliminated from the germ line genome during development of the somatic macronucleus. They are flanked by one 5'-TA-3' dinucleotide on each side, a single copy of which remains at the donor site after excision. The timing of their excision was examined in synchronized conjugating cells by quantitative PCR. Significant amplification of the germ line genome was observed prior to IES excision, which starts 12 to 14 h after initiation of conjugation and extends over a 2- to 4-h period. Following excision, two IESs were shown to form extrachromosomal circles that can be readily detected on Southern blots of genomic DNA from cells undergoing macronuclear development. On these circular molecules, covalently joined IES ends are separated by one copy of the flanking 5'-TA-3' repeat. The similar structures of the junctions formed on the excised and donor molecules point to a central role for this dinucleotide in IES excision.

Animals↗
PubMed2004

RNA-mediated programming of developmental genome rearrangements in Paramecium tetraurelia.

The germ line genome of ciliates is extensively rearranged during development of the somatic macronucleus. Numerous sequences are eliminated, while others are amplified to a high ploidy level. In the Paramecium aurelia group of species, transformation of the maternal macronucleus with transgenes at high copy numbers can induce the deletion of homologous genes in sexual progeny, when a new macronucleus develops from the wild-type germ line. We show that this trans-nuclear effect correlates with homology-dependent silencing of maternal genes before autogamy and with the accumulation of approximately 22- to 23-nucleotide (nt) RNA molecules. The same effects are induced by feeding cells before meiosis with bacteria containing double-stranded RNA, suggesting that small interfering RNA-like molecules can target deletions. Furthermore, experimentally induced macronuclear deletions are spontaneously reproduced in subsequent sexual generations, and reintroduction of the missing gene into the variant macronucleus restores developmental amplification in sexual progeny. We discuss the possible roles of the approximately 22- to 23-nt RNAs in the targeting of deletions and the implications for the RNA-mediated genome-scanning process that is thought to determine developmentally regulated rearrangements in ciliates.

Animals↗
PubMed1983

Structure and expression of genes for surface proteins in Paramecium.

Surface proteins from 11 antigenic types of Paramecium tetraurelia vary in molecular weight from 251,000 to 308,000. The size of a series of polyadenylated RNAs obtained from these types were correlated with the sizes of the proteins and judged to be the mRNAs for the proteins. The mRNAs were used to identify genomic DNA clones containing complementary sequences. The gene for antigen A was present in one copy per genome, and the data suggest that extensive introns were absent. When restriction enzyme digests of DNA from cultures of paramecia with active and inactive genes were probed with portions of the cloned genes, no evidence for rearrangements or changes in gene dosage was found.

Animals↗
PubMed1986

Induced change in a non-mendelian determinant by transplantation of macronucleoplasm in Paramecium tetraurelia.

Mutant strain d48 of Paramecium tetraurelia lacks the gene for antigen A in the macronucleus, whereas this gene is present in the micronucleus. Transfer of macronucleoplasm from the wild type to strain d48 caused d48 to revert to the wild type after autogamy. Transfer of cytoplasm was not as effective as transfer of macronucleoplasm. It was also found that the micronucleus of d48 developed normally when it was transplanted to wild-type cells, whereas the micronucleus of the wild type formed a macronucleus that lacked the antigen A gene when this micronucleus was transplanted into d48. It was concluded that the micronucleus of d48 has a normal antigen A gene and that the hereditary determinants responsible for the d48 trait are located in the macronucleus. Molecular analysis of d48 clones that had been induced to revert to the wild type revealed that they possessed the antigen A gene in the macronucleus.

Animals↗