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.     

Structure of mitochondrial DNA from Paramecium tetraurelia

Mitochondrial DNA (mtDNA) from endosymbiote-free stocks of Paramecium tetraurelia was isolated by 2 procedures. The buoyant density of the mtDNA in neutral CsCl was 1.702 gm/cm3, a value consistent with the melting temperature of the mtDNA. Only linear molecules were observed by electron microscopy. These molecules were homogeneous in size with a monomer molecular weight of 25.6 x 10(6) daltons. The size of the mtDNA determined after digestion with the restriction endonucleases EcoRI or Hind III agreed with the value obtained by electron microscopy. These studies also revealed that the digestion pattern of mtDNA from stock 172 differed from that of other 3 stocks (51, 127, 203) examined. Some mtDNA molecules exhibited snapback reassociation following denaturation.     

Responses to hypergravity in proliferation of Paramecium tetraurelia

It has been reported that Paramecium proliferates faster when cultured under microgravity in orbit, and slower when cultured under hypergravity. This shows that the proliferation rate of Paramecium affected by gravity. The effect of gravity on Paramecium proliferation has been argued to be direct in a paper with an axenic culture under hypergravity. To clear up uncertainties with regard to the effect of gravity, Paramecium tetraurelia was cultured axenically under hypergravity (20 x g) and the time course of the proliferation was investigated quantitatively by a new non-invasive method, laser-beam optical slice, for measuring the cell density. This method includes optical slicing a part of the culture and computer-aided counting of cells in the sliced volume. The effects of hypergravity were assessed by comparing the kinetic parameters of proliferation that were obtained through a numerical analysis based on the logistic growth equation. Cells grown under 20 x g conditions had a significantly lower proliferation rate, and had a lower population density at the stationary phase. The lowered proliferation rate continued as long as cells were exposed to hypergravity (> one month). Hypergravity reduced the cell size of Paramecium. The long and short axes of the cell became shorter at 20 x g than those of control cells, which indicates a decrease in volume of the cell grown under hypergravity and is consistent with the reported increase in cell volume under microgravity. The reduced proliferation rate implies changes in biological time defined by fission age. In fact the length of autogamy immaturity decreased by measure of clock time, whereas it remained unchanged by measure of fission age.     

转基因植物转录后基因沉默的克服对策

人们对植物进行遗传转化的目的是让转基因在植物基因组中能够稳定整合并在当代及其子代中能够有效、稳定的表达 ,但是由于多种因素的影响 ,转基因在植物中的表达并不很理想。自从 1 986年Ｐｅｅｒｂｏｔｔｅ报道转基因烟草中转基因发生沉默以来 ,很多学者也都发现大量的转基因植株不能正常表达[1]。经过多年的研究 ,发现导致转基因沉默的机理有多种 ,根据其作用机理和水平的不同 ,人们通常把转基因沉默分为[2 ,3]:位置依赖性基因沉默 (Ｐｏｓｔｉｏｎ -ｄｅｐｅｎｄｅｎｔｇｅｎｅｓｉｌｅｎｃｉｎｇ ,ＰＤＧＳ)、转录水平的基因沉默 (Ｔｒ…     

Organization of ribosomal genes in Paramecium tetraurelia

The macronuclear ribosomal DNA (rDNA) of the ciliated protozoan Paramecium tetraurelia (stock 51) was analyzed by digestion with restriction endonucleases. The fragments which contained ribosomal RNA (rRNA) coding sequences and spacer sequences were identified. The spacer sequences exhibited some heterogeneity in size. The genes coding for 5.8S RNA, but not for 5S RNA, are linked to the 17S and 25S rRNA genes. Complementary RNA, synthesized from rDNA of stock 51, was hybridized with restriction digests of whole cell DNA from six other allopatric stocks of this species. The restriction patterns of the rDNA from these seven stocks were, in general, very similar, and the sizes of the coding sequences were identical in all seven stocks. Only the restriction pattern of rDNA from stock 127 differed significantly from that of stock 51. The rDNA from stock 127 was isolated and characterized, and with the exception of the restriction pattern of its spacer, it resembled the rDNA from stock 51. It is concluded that the rDNA repeat in Paramecium, including the spacer, has, in general, been conserved during the course of evolution. It is suggested that in some species, even in the absence of genetic exchange among geographically separated populations, selection pressure may act to conserve spacers of tandemly repeated rDNA. The conservation may be related to the number of rDNA copies in the germinal nucleus.     

A new multigene family encoding calcium-dependent calmodulin-binding membrane proteins of Paramecium tetraurelia

Ca2+/calmodulin (CaM) regulates various physiological processes in a wide variety of organisms, metazoa and protists alike. To better understand Ca2+/CaM-dependent processes, particularly those with membrane-associated components, we studied Ca2+/CaM-binding membrane proteins in Paramecium tetraurelia, a unicellular model system. A CaM-binding protein, PCM1 (Paramecium CaM-binding membrane-bound protein), from a detergent-solubilized ciliary membrane fraction was identified and purified through Ca2+-dependent CaM-affinity chromatography. PCM1 has an apparent molecular mass of approx. 65kDa. It binds radiolabeled CaM in blot overlay assays and binds to CaM-affinity columns, both only in the presence of 10 microM or higher Ca2+. Three peptide sequences from PCM1 were obtained, and polymerase chain reaction (PCR) and Southern hybridization experiments were designed accordingly, leading to a partial cDNA clone for PCM1 and the discovery of three homologs: PCM2, PCM3 and PCM4. Amino acid sequences predicted by the full-length coding sequence for PCM3 and partial genes for PCM1, PCM2 and PCM4 are very similar (approx. 85% amino-acid identities). Their sequences indicate that they are hitherto novel proteins with beta/gamma-crystallin domains, cysteine-rich regions and potential CaM-binding domains. These protein motifs are suggested to mediate protein-protein interaction important for Ca2+/CaM signal transduction event(s) through the PCM family of proteins.     

Chromogranin A-like proteins in the secretory granules of a protozoan, Paramecium tetraurelia

The ciliate protozoan Paramecium tetraurelia produces secretory granules (trichocysts) which release needle-like structures composed of small, acidic proteins. Using antibodies against isolated chromogranin A (CGA) and against trichocyst proteins, we found cross-reactive proteins in chromaffin granules and trichocysts. Four independently derived sera against isolated CGA stained bands of the Mr 15,000-25,000 family of trichocyst proteins on immunoblots. A positive response was also obtained with antiserum against chemically synthesized peptides (PL26 and GE25) corresponding to defined regions of the CGA amino acid sequence. In extracts of whole Paramecium, larger proteins (Mr 53,000 and 49,000) also reacted with antibodies against CGA and the related synthetic peptides. These larger proteins may represent unprocessed precursors to the smaller proteins of mature trichocysts. Antiserum to trichocysts recognized CGA in chromaffin granule lysates. Further evidence of a Paramecium protein related to CGA was provided by hybridization of Paramecium mRNA with cloned cDNA for bovine CGA. Our results suggest striking conservation in evolution of CGA-like proteins that may play some role, as yet unknown, in secretion.     

Purification and characterization of a calcium-dependent ATPase from Paramecium tetraurelia

We report the purification of a CaATPase of high specific activity from Paramecium tetraurelia. The enzyme is preferentially released into solution upon deciliation of cells by a Ca2+ shock procedure. Purification by ion exchange and gel filtration chromatography yields major peptides of 68 and 53 kDa and a minor peptide of 58 kDa, as determined by electrophoresis on sodium dodecyl sulfate polyacrylamide gels. These three peptides yield similar proteolytic peptide maps. Rabbit antisera to the purified enzyme inhibit enzyme activity and specifically label 68- and 53-kDa bands on nitrocellulose blots of the deciliation supernatant from which the enzyme is isolated. Concanavalin A-Sepharose precipitates about 60% of ATPase activity; only the 53-kDa band binds concanavalin A on nitrocellulose blots. The purified enzyme has a specific activity of 620 +/- 70 mumol/min/mg with ATP as substrate in the presence of Ca2+, which is required for enzyme activity. As substrates, ATP and GTP are strongly preferred to UTP and CTP. The Km for ATP in the presence of 3 mM Ca2+ is approximately 20 microM. Enzyme activity is strongly inhibited by the calmodulin antagonists trifluoperazine, fluphenazine, W7, and calmidazolium. However, calmodulin is not associated with the purified enzyme, based on the enzyme's inability to bind anti-calmodulin antibodies or to stimulate brain phosphodiesterase. The intracellular origin of this ATPase, its possible function, and its relationship to several other ATPases of Paramecium are discussed.     

Non-Mendelian inheritance induced by gene amplification in the germ nucleus of Paramecium tetraurelia

A genetic investigation of strain d4-95, which carries a recessive mutant allele (pwB(95)) of pawn-B, one of the controlling elements of voltage-dependent calcium channels in Paramecium tetraurelia, revealed a non-Mendelian feature. Progeny of the cross between d4-95 and wild type often expressed a clonally stable mutant phenotype, even when they had a wild-type gene. The mutant phenotype was also expressed after self-fertilization of theoretical wild-type homozygotes recovered from the cross. Our molecular analysis demonstrated that the copy number of the mutant pwB gene in the micro- and macronucleus of d4-95 was much greater than that of the wild type. Most of the amplified, extra pwB gene copies in d4-95 were heritable independently from the original pwB locus. Repeated backcrossing of d4-95 with the wild type to dilute extra pwB genes in the strain produced segregants with a completely normal Mendelian trait in testcrosses. These results strongly suggest that a non-Mendelian inheritance of d4-95 was induced by gene amplification in the micronucleus.     

Cysteine residue periodicity is a conserved structural feature of variable surface proteins from Paramecium tetraurelia.

The DNA sequences of the entire coding regions of the A and C type variable surface protein genes from Paramecium tetraurelia, stock 51 have been determined. The 8151 nucleotide open reading frame of the A gene contains several tandem repeats of 210 nucleotides within the central portion of the molecule as well as a periodic structure defined by cysteine residues. The 6699 nucleotide open reading frame of the C gene does not contain any identifiable tandem repeats or internal similarity but maintains a periodicity based on the cysteine residue spacing. The deduced amino acid sequences encoded by the two genes are most similar within the 600 amino-terminal and 600 carboxyl-terminal amino acid residues, the central portions show only limited sequence similarity. We conclude that internal repeats are not a conserved feature of variable surface proteins in Paramecium and discuss the possible importance of the regular pattern of cysteine residues.     

Micronuclear DNA from Paramecium tetraurelia: serotype 51 A gene has internally eliminated sequences.

A method for the isolation of micronuclear DNA from Paramecium tetraurelia has been developed. After cell lysis, a low speed centrifugation at 1,000 g is used to remove all of the unbroken cells and macronuclei and approximately two thirds of the macronuclear fragments. Next a higher speed centrifugation of 9,000 g sediments the micronuclei and frees them from small particulates and soluble constituents. Advantage is then taken of the fact that micronuclei have a lower density than do macronuclear fragments in 45%-60% Percoll. Micronuclei float to the top during centrifugation at 24,000 g, while macronuclear fragments sediment. After several cycles of centrifugation in Percoll, the micronuclei, although heavily contaminated with cytoplasmic components, are essentially free of macronuclei and macronuclear fragments. Micronuclear DNA can then be extracted from the suspension. The whole procedure is very rapid and in about an hour micronuclear and macronuclear DNA can be separated. About 2 micrograms of micronuclear DNA can be obtained from 6 x 10(7) paramecia. We find that there are internal sequences in the micronuclear A gene DNA in wild type cells which are eliminated when the micronuclei develop into macronuclei. They yield unique restriction fragments for micronuclei and macronuclei. Therefore the purity of the preparations is easily monitored by probing Southern blots of restriction enzyme-digested DNA with the cloned A gene. No differences have been found between the micronuclear A gene in wild type and the d48 mutant.     

Accumulation of DNA damages in aging Paramecium tetraurelia

Summary Paramecium tetraurelia cells of ages 4, 15, and 27 days were labeled with [¹⁴C]-thymidine. In addition, cells were grown clonally for 27 days (108 generations) and labeled with [¹⁴C]-thymidine in the presence of 0.5 or 7.5 g/ml of mitomycin-C (MMC) or no MMC. These cells were gently deposited on a filter membrane, which impedes the passage of DNA strands. The cells were then lysed with detergents and the cellular components washed through the filters, leaving double-stranded DNA intact on the surface. Proteinase K was used to remove histone or DNA-bound proteins. The DNA was then eluted under alkaline conditions, which denatures double-stranded DNA and converts apurinic/apyrimidinic sites into single-strand breaks. The results obtained with the cells of ages 4, 15, and 27 days (16, 60, and 108 generations, respectively) indicate that as Paramecium tetraurelia ages during asexual reproduction, apurinic/apyrimidinic lesions, strand breaks or single-strand gaps accumulate. This accumulation may be the basic mechanism of aging in such cells. In the MMC-treated cells of 27 days (108 generations), the MMC reduced elution of DNA fragments more at the higher than at the lower pH's used; random MMC cross-links should occur more often in longer strands than in shorter strands. The reductions in elution preferentially at higher pH, at which longer single strands would be eluted, confirmed the pH-versuslength relationship for Paramecium DNA eluted under our conditions.     

Calmodulin in Paramecium tetraurelia: localization from the in vivo to the ultrastructural level

Monospecific polyclonal antibodies against Paramecium tetraurelia calmodulin were prepared and labeled for calmodulin localization on different levels of resolution: by microinjection into living cells; with isolated cell surface complexes (cortices); on the ultrastructural level, using Lowicryl sections of non-permeabilized cells (with colloidal gold-protein A labeling of antibodies bound); or using permeabilized and gently fixed cells for incubation with peroxidase- or microperoxidase-tagged antibodies. Sites selectively labeled above cytoplasmic background largely coincided, irrespective of the method used, although sensitivity, resolution, and liability to redistribution of antigen were quite different. (The methodological diversification applied allowed for their mutual control.) Nonspecific binding can be largely excluded, since all these methods gave negative results with pre-immune sera. We reached the following conclusions on sites with selective calmodulin binding (above cytoplasmic background level) in P. tetraurelia cells. A pool of calmodulin co-localized with F-actin, not only in the cortex (including fibrous materials around ciliary basal bodies) but also around food vacuoles (phagosomes) and, to a lesser degree, around the buccal cavity. Trichocyst docking sites on the cell membrane, and coated pits also displayed calmodulin labeling, thus indicating the potential involvement of calmodulin in exo-endocytosis processes. Calmodulin was also enriched on membranes of compartments with presumable ion (possibly Ca2+) transport capacity, such as trichocysts and the osmoregulatory system. Not selectively labeled were nuclei, mitochondria, and some small lysosomal organelles (as identified in vivo by rhodamine 123 or acridine orange fluorescence, respectively).