Genome-defence small RNAs exapted for epigenetic mating-type inheritance.

In the ciliate Paramecium, transposable elements and their single-copy remnants are deleted during the development of somatic macronuclei from germline micronuclei, at each sexual generation. Deletions are targeted by scnRNAs, small RNAs produced from the germ line during meiosis that first scan the maternal macronuclear genome to identify missing sequences, and then allow the zygotic macronucleus to reproduce the same deletions. Here we show that this process accounts for the maternal inheritance of mating types in Paramecium tetraurelia, a long-standing problem in epigenetics. Mating type E depends on expression of the transmembrane protein mtA, and the default type O is determined during development by scnRNA-dependent excision of the mtA promoter. In the sibling species Paramecium septaurelia, mating type O is determined by coding-sequence deletions in a different gene, mtB, which is specifically required for mtA expression. These independently evolved mechanisms suggest frequent exaptation of the scnRNA pathway to regulate cellular genes and mediate transgenerational epigenetic inheritance of essential phenotypic polymorphisms.

The Paramecium germline genome provides a niche for intragenic parasitic DNA: evolutionary dynamics of internal eliminated sequences.

Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events. In Paramecium, these rearrangements include precise excision of unique-copy Internal Eliminated Sequences (IES) from the somatic DNA, requiring the activity of a domesticated piggyBac transposase, PiggyMac. We have sequenced Paramecium tetraurelia germline DNA, establishing a genome-wide catalogue of -45,000 IESs, in order to gain insight into their evolutionary origin and excision mechanism. We obtained direct evidence that PiggyMac is required for excision of all IESs. Homology with known P. tetraurelia Tc1/mariner transposons, described here, indicates that at least a fraction of IESs derive from these elements. Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P. aurelia species complex, but IES invasion of the Paramecium genome appears to be an ongoing process. Once inserted, IESs decay rapidly by accumulation of deletions and point substitutions. Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a -10 bp periodicity, corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex. IESs are equally frequent within and between coding sequences; however, excision is not 100% efficient and there is selective pressure against IES insertions, in particular within highly expressed genes. We discuss the possibility that ancient domestication of a piggyBac transposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences, a fraction of the genome in which parasitic DNA is not usually tolerated.

New Stands of Species of the Paramecium aurelia Complex; is the Occurrence of Particular Species Limited by Temperature Barriers?

The occurrence of ciliates, especially the Paramecium aurelia complex, has not yet been studied in many parts of the world, or sampling was done only occasionally. Generally, the southern hemisphere still awaits investigation. In North America only the USA was studied in greater detail; the majority of species of the complex were there recorded. In Asia, more frequent sampling was performed only in Japan and Asiatic Russia. Europe was studied more carefully, however, a different number of habitats was studied in particular zones of Europe, the least in the southern zone. New stands of P. tetraurelia , P. sexaurelia, P. octaurelia, and P. novaurelia were revealed as a result of the present investigations carried out in Africa (Mozambique--P. tetraurelia, P. sexaurelia), Asia (Indonesia--P. sexaurelia), borderland of Asia and Europe (Georgia--P. octaurelia), and Europe (Macedonia--P. tetraurelia and Romania--P. novaurelia). Are climatic zones the main factor limiting the occurrence of species of the P. aurelia complex? Analysis of data on the distribution of the P. aurelia species complex in warm "tropical" zones on different continents may suggest such preferences for some species, including P. sexaurelia, P. octaurelia, P. tredecaurelia, P. quadecaurelia. The first two of these species were recorded herein in warm or "tropical" zone.

The ts111 Mutation of Paramecium tetraurelia Affects a Member of the Protein Palmitoylation Family.

The thermosensitive ts111 mutant of Parameciun tetraurelia carries a recessive mutation which causes cell death after 2-8 divisions at the restrictive temperature of 35 degrees C. Expression at 35 degrees C induces disassembly of the infraciliary lattice (ICL). In this study, we found that the ts111 mutation also results in significant abnormalities in the number and structure of contractile vacuole complexes (CVCs) and in their functioning at the restrictive temperature. In order to characterize the ts111 gene, the complementation cloning was performed by microinjection into the macronucleus of an indexed genomic DNA library. The mutation was complemented by a sequence of 852 bp, which differed from the mutant sequence by a single nucleotide substitution. The deduced protein sequence is 284 amino acids long. It contains a domain referred to as the DHHC domain, associated with 2 trans-membrane helices. The DHHC proteins belong to the Palmitoyl-Acyl Transferases (PATs) protein family, which is implicated in the protein palmitoylation process playing the role in protein addressing. The ts111 mutation induces the amino acid change, localized before the first membrane helix. Transformation of ts111 mutant cells with the TS111-GFP gene fusion showed the expected reparation restoring thermoresistance and also demonstrated a localization of the protein in contractile vacuoles, but not in the ICL. The entire gene silencing in wild type cells at restrictive temperature caused the same effect as the expression of a point mutation in ts111 mutant. The authors propose the following hypotheses: (i) function of CVCs at the restrictive temperature depends in Paramecium on the TS111 protein--a member of the PAT family, and the primary effect of the termosensitive ts111 mutation are morphological abnormalities and dysfunction of CVCs, (ii) disassembly of the ICL is a secondary effect of the ts111 mutation, which results from disturbed regulation of the intracellular concentration of Ca(+2) ions caused by the abnormal functioning of CVC.

Genetic and epigenetic factors affecting meiosis induction in eukaryotes revealed in paramecium research.

This review presents studies of the induction of meiosis undertaken on the ciliate Paramecium, a unicellular model eukaryotic organism. Meiosis in Paramecium, preceding the process of fertilization, appears in starved cells after passing a defined number of divisions (cell generations), starting from the last fertilization. Investigations were performed on clones of cells entering autogamy, a self-fertilization process. Genetic as well as epigenetic factors, i.e. endo- and exogenous factors, affecting the induction ofmeiosis and changing the duration of the interautogamous interval (IAI), were analyzed. The results show that: (1) Meiosis induction is controlled genetically by the somatic macronucleus. However, besides the nuclear factors, the cytoplasmic protein immaturin also affects this process (Haga & Hiwatashi 1981); (2) Epigenetic factors, such as non-genetically disturbed cytoskeleton structures and changes in the cell architecture observed in doublet Paramecium cells, exert internal mechanical stress (Ingber 2003), which constitutes the endogenous impulse accelerating meiosis; (3) Mild osmotic stress, acting as an exogenous factor, can initiate the specific MAP kinases signaling pathway resulting in earlier meiosis induction, as in other unicellular eukaryotes (Seet & Pawson 2004).

Glucose, sorbitol and insulin as exogenous agents that monitor the interautogamous interval (IAI) in Paramecium primaurelia.

Results from previous studies have revealed that the duration of the interautogamous interval (IAI), expressed as the number of fissions occurring between two successive autogamies, is stable and characteristic for species of the Paramecium aurelia complex (Kósciuszko & Prajer 1988; Prajer & Kosciuszko 1999). The duration of the IAI is genetically controlled (Kosciuszko & Prajer 1989; Mikami & Koizumi 1983). Genomic mutations (Prajer & Kosciuszko 1999; Takagi et al. 1989) as well as disturbances of morphological homeostasis of the cell, as observed in Paramecium doublets (Prajer et al. 1999), shorten the duration of the IAI. This suggests that epigenetic factors may accelerate the induction of autogamy. The aim of this study was to search for environmental, exogenous factors which may affect the IAI duration. The first investigated factor was glucose used in such a concentration which did not change the rate of vegetative cell divisions. The second tested factor was sorbitol used as nonmetabolized sugar and applied at a similar concentration as glucose to verify a possible osmotic effect. Both these factors accelerated the induction of autogamy and shortened IAI duration in Paramecium primaurelia. An insulin hormone was also used as a candidate for a sugar dependent factor complementary to glucose. It also resulted in acceleration of autogamy induction. It was chosen to test its effect on IAI since the existence of an insulin receptor in Tetrahymena has been reported (Christensen et al. 2003). This hormone peptide also induced an IAI shortening. The results of a search in the Ciliates genome for potential homologs of genes coding insulin receptors and insulin itself, as well as hypothetical mechanisms of action of investigated agents were discussed.

Molecular analysis (RAPD-PCR) of inter-strain hybrids of the Paramecium aurelia species complex (Ciliophora, Protozoa).

RAPD-PCR analysis showed that species of the Paramecium aurelia complex possessed characteristic band patterns and that the majority were also polymorphic intra-specifically. A comparison of band patterns was performed for some inter-strain hybrids within P. primaurelia, P. tetraurelia, P. pentaurelia, P. septaurelia, P. octaurelia, P. decaurelia, P. dodecaurelia, P. tredecaurelia, and P. quadecaurelia to band patterns characteristic for the parental strains. The investigations, however, did not reveal a close correlation between the degree of inbreeding characteristic for the species and similarity of genotypes. A low similarity of hybrid and parental band patterns was observed in P. octaurelia, P. dodecaurelia, P. quadecaurelia and also P. primaurelia. A high similarity of band patterns of hybrid and parental strains was found in P. tetraurelia, P. septaurelia, P. decaurelia, and P. tredecaurelia.

New European stands of Paramecium pentaurelia, Paramecium septaurelia, and Paramecium dodecaurelia, genetic and molecular studies.

New stands of rare species of the Paramecium aurelia complex were found in Europe, i.e. P. pentaurelia and P. dodecaurelia in Italy and P. septaurelia in Germany. The species were identified by mating reactions with the standard strains of each species. Their relationships with some other known strains of particular species were studied by classical strain crosses (survival in F1 and F2 generations) and by comparison of RAPD-PCR fingerprints. The presence of the cosmopolitan species P. tetraurelia in Italy was also recorded.

A two-locus molecular characterization of Paramecium calkinsi.

Paramecium calkinsi (Ciliophora, Protozoa) is a euryhaline species which was first identified in freshwater habitats, but subsequently several strains were also collected from brackish water. It is characterized by clockwise spiral swimming movement and the general morphology of the "bursaria type." The present paper is the first molecular characterization of P. calkinsi strains recently collected in distant regions in Russia using ITS1-5.8S- ITS2-5'LSU rDNA (1100bp) and COI (620bp) mtDNA sequenced gene fragments. For comparison, our molecular analysis includes P. bursaria, exhibiting a similar "bursaria morphotype" as well as species representing the "aurelia type," i.e., P. caudatum, P. multimicronucleatum, P. jenningsi, and P. schewiakoffi, and some species of the P. aurelia species complex (P. primaurelia, P. tetraurelia, P. sexaurelia, and P. tredecaurelia). We also use data from GenBank concerning other species in the genus Paramecium and Tetrahymena (which used as an outgroup). The division of the genus Paramecium into four subgenera (proposed by Fokin et al. 2004) is clearly presented by the trees. There is a clear separation between P. calkinsi strains collected from different regions (races). Consequently, given the molecular distances between them, it seems that these races may represent different syngens within the species.