Targeted gene knockout of inner arm 1 in Tetrahymena thermophila

Cilia and flagella contain at least eight different types of dynein arms. It is not entirely clear how the different types of arms are organized along the axoneme. In addition, the role each different type of dynein plays in ciliary or flagellar motility is not known. To initiate studies of dynein organization and function in cilia, we have introduced a mutation into one dynein heavy chain gene (DYH6) in Tetrahymena themophila by targeted gene knockout. We have generated mutant cells that lack wild-type copies of the DYH6 gene. We have shown that the DYH6 gene encodes one heavy chain (HC2) of Tetrahymena 18S dynein and that 18S dynein occupies the I1 position in the ciliary axoneme. We have also shown that Tetrahymena I1 is required for normal motility, normal feeding and normal doubling rate.     

Chemosensory behaviour and ciliary cyclic GMP-dependent protein kinase in Tetrahymena thermophila

The role of protein kinase, in particular cyclic GMP-dependent protein kinase (PKG), in the control of chemotaxis was studied in Tetrahymena thermophila using the membrane-permeable cGMP analogue 8-bromo-cGMP and the NO-generator sodium nitroprusside (SNP) that stimulates cGMP production by activating guanylate cyclase. Stimulation of chemoattraction was observed in the presence of 8-bromo-cGMP and nitroprusside when used in 10–100 μM concentrations in vivo. In vitro stimulation of ciliary membrane PKG activity was observed when using similar concentrations of cGMP or 8-bromo-cGMP to those in the in vivo experiments. In contrast, the protein kinase flavonol inhibitors quercitin and kaempherol block chemoattraction and reduce ciliary membrane PGK activity in vitro. For the inhibition of PKG, the IC-50 s for quercitin and kaempherol are 22 and 19 μM, respectively. The results suggest a modulating function of PKG on adaptory processes in cilia-mediated chemotaxis.

The ciliary membrane-associated PKG was partially characterized. Without added external protein kinase substrate in vitro, an endogenous ciliary membrane kinase activity showed phosphorylation of 55 and 97 kDa Triton-X-100 soluble proteins when analyzed by SDS-PAGE under reducing conditions and with ³²P-γ-ATP as phosphorylation donor. Phosphoamino acid analysis of PKG-phosphorylated proteins showed ³²P-phosphate labeling of serine and threonine residues. Ciliary membrane-associated PKG was further purified by carboxy-methyl-sephadex-column chromatography. The membrane enzyme was Mg²⁺⁺-dependent and had a pH optimum at 6.4. The carboxy-methyl-sephadex-eluted PKG was analyzed by electrophoresis on sodium dodecyl sulphate polyacrylamide gels showing a molecular weight of 70–75 kDa.     

An amicronucleate mutant of Tetrahymena thermophila

A stable amicronucleate strain of Tetrahymena thermophila was isolated following nitrosoguanidine mutagenesis. The mutant has the same growth rate and viability as the micronucleate parent strain, and has no micronucleus detectable by chromatin-specific staining in vegetative growth or during conjugation. The mutant pairs with normal efficiency with cells of complementary mating type. Matings of the mutant with aneuploid strains which lose their micronucleus during meiosis produced cell pairs yielding one viable and one inviable cell. The mutant receives a micronucleus from a normal mating partner, but this micronucleus is lost by the mutant cells within two hundred generations.     

Protein synthesis patterns in conjugating Tetrahymena thermophila

Conjugation in Tetrahymena is a developmental system which results in a synchronized reorganization of the genetic material in both mates of a pair. Mass cultures of T. thermophila are easily induced to conjugate simultaneously, thus conjugation is amenable to cytological, genetical and biochemical studies. We investigated the changes in newly synthesized whole-cell proteins as a function of time after mixing of mating-competent cells, using [³⁵S]methionine pulse-labelling, one-dimensional polyacrylamide gel electrophoresis, and autoradiography. Concomitantly, the percentage of pairs and the cytological events were followed. Synthesis of at least ten protein bands with molecular weights larger than 50 kD are stimulated during the 5 h interval between mixing of the cells and meiosis of the gametic nuclei, whereas only minor protein synthesis takes place in the following 3 h. Two bands in particular are stimulated: one (81 kD, actD-insensitive) is synthesized until cells pair while the other (86 kD, actD-sensitive) is found between pairing and meiosis only.     

Polypeptides during early conjugation in Tetrahymena thermophila

As Tetrahymena thermophila cells differentiate from their vegetative life cycle to sexual reproduction, their polypeptide pattern undergoes a series of changes. These changes have been traced in extracellular, cellular, and subcellular compartments. The first alteration is induced by the nutritional shift-down and results in stimulation of at least one ciliary polypeptide and affects a series of polypeptides from other compartments. The second alteration is induced by mixing starved cells of complementary mating types and this stimulates the synthesis of nine ciliary polypeptides before pairs have formed and eight afterwards. At least five of these early and one of the late conjugation-related ciliary polypeptides are removed by low concentrations of EDTA, indicating that they are located on the external side of the plasma membrane. No differences were observed between polypeptides excreted during starvation and after mixing of complementary mating types. At Tris concentrations restrictive for conjugation, cilia lack the conjugation-related polypeptides. Some of these are instead found among the excreted polypeptides. Using O'Farrell gels and silver staining on isogenic cells of all possible mating types, we have been unable to correlate changes in polypeptide patterns to specific mating types.     

A Transmissible developmental block in Tetrahymena thermophila

When the amicronucleate mutant BI3840 of Tetrahymena thermophila is mated with normal micronucleate cells, it receives a pronucleus from its partner but there is no further nuclear development and the conjugants separate, retaining their original macronuclei. Both of these sexually mature exconjugants and any cells with which they are mated show an unconditional block in macronuclear development. Although prezygotic nuclear divisions, nuclear transfer and post-zygotic nuclear divisions appeared normal upon cytological analysis of Giemsa-stained conjugants, macronuclear development was invariably aborted. Since the original macronucleus was resorbed, the cells were rendered amacronucleate and they died. When no macronuclear development was initiated, as in crosses with the aneuploid strain A* (III), the exconjugants were viable and retained their original macronuclei. This pattern was invariant with three different strains serving as the original micronuclear source, and in the case of one non-BI3840 exconjugant, persisted for over 200 cell generations. Exconjugants from a cross of one of the micronuclear donors with strain A* (III) did not show arrested development when crossed. It thus appears likely that there is conjugal transfer of non-nuclear information originating in BI3840 which is self-replicating and which causes an arrest in macronuclear development.     

Autoradiographic evidence for self-fertilization in Tetrahymena thermophila

Conjugation in Tetrahymena thermophila consists of a sequence of nuclear events, including meiosis and reciprocal cross-fertilization, which result in biparental genetic endowment of the sexual progeny. Genetic evidence was recently provided that the normal exchange of gametic nuclei between conjugating cells can be efficiently blocked by hyperosmotic shock. In this paper we confirm this finding autoradiographically. We also report that the inhibitor of microtubule assembly, vinblastine, also blocks this step, as well as the subsequent fusion of gametic nuclei. The ability of conjugating cells to survive and continue more or less normally after blocks of self-fertilization and pro-nuclear fusion demonstrates a surprisingly high degree of developmental regulation during conjugation. Self-fertilization has proven useful for the isolation of recessive mutants in T. thermophila.     

ConcanavalinA receptors and the chemosensory behaviour of Tetrahymena thermophila

The relationship between concanavalin A (ConA) receptors and the chemosensory behaviour of the ciliated protozoan Tetrahymena thermophila was studied using the peptide chemoattractants proteose peptone and fibroblast growth factor. Studies on the chemosensory behaviour in semisolid methylcellulose showed that 50 μg/ml ConA selectively inhibited the persistent element of swimming behaviour by reducing time runs of cells responding to proteose peptone from 12.2±4.5 min to 0.8±0.3 min. Methyl-alpha-D-mannoside, but not methyl-alpha-D-galactoside, abolished the inhibitory effect of ConA, suggesting that mannoside-containing ConA receptors are involved in maintaining a persistent swimming behaviour. Control experiments, carried out in liquids where persistent swimming is less important for cellular behaviour, showed that ConA did not affect proteose-peptone-induced chemoattraction under these conditions as measured by a two-phase assay for chemoattraction. Also, no inhibitory effect of ConA could be found on swimming rates when individual velocities of ConA-treated cells were determined. When tested in liquid chemoattraction assays, ConA was found to be a weak but significant chemoattractant. Studies of the cellular location of ConA receptors on the plasma membrane of starved cells showed an unequal distribution. A preferential clustering of receptors at the anterior end of the cell was observed when determined at high concentrations (100 μg/ml) of fluorescent ConA. Methyl-alpha-D-mannoside but not methyl-alpha-D-galactoside abolished the fluorescent ConA labelling, indicating a preferential clustering of these mannoside-containing receptors at the anterior part of the plasma membrane and cilia. At lower concentrations (25 μg/ml), FITC-ConA produced more general labelling of the entire cell membrane. The results suggest that ConA receptors are necessary for the persistent element of swimming and that binding of ConA to its receptors interferes with processes related to signal transduction rather than by limiting the free movement of cilia required for locomotion. The gradient of receptors seen at high FITC-ConA concentrations may be important for a putative spatial chemosensory mechanism, i.e. chemotaxis.Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Stage-specific changes in protein synthesis during conjugation in Tetrahymena thermophila

Conjugation in the free-living ciliate Tetrahymena thermophila is an inducible developmental system which results in a synchronized reorganization of the genetic material in both mates of a pair. The cytological events were followed by Feulgen stainings of simultaneously mating cells and protein synthesis was revealed using [³⁵S]methionine pulse labelling and two-dimensional gel electrophoresis. At least 33 proteins, including 24 conjugation-specific proteins, with apparent molecular weights (M_r) between 61 and 200 × 10³ are stimulated during conjugation. Two slightly acidic proteins (M_r 89 and 73 × 10³, respectively) are stimulated shortly after mixing of mating-competent cells and mainly before tight pairs are formed. Ten proteins are stimulated during meiosis, and two of these (M_r 90 and 78 × 10³, respectively) are particularly interesting, since they are highly stimulated and more basic (pI values around 8.5) than most other proteins detected. Twelve proteins are stimulated essentially between pairing and early macronuclear development, three are stimulated from shortly before zygote formation and during the postzygotic divisions, and six are stimulated during late conjugation, at various parts of macronuclear development. The functions of the conjugation-stimulated proteins are discussed.     

Transformation of Tetrahymena thermophila by electroporation and parameters effecting cell survival

We have successfully transformed Tetrahymena thermophila by electroporation, a process of electrically introducing DNA. The DNA used for transformation contains a mutant ribosomal RNA gene (rDNA) that confers resistance to paromomycin on the transformed cells. This mutant rDNA replicates more rapidly than the endogenous rDNA of the transformed cells so that the mutant rDNA becomes predominant within several generations. This mutant rDNA also carries a restriction polymorphism that readily distinguishes it from the endogenous rDNA of the transformed cells. Substantial nuclease activity is released from the cells during electroporation and must be neutralized in order for transformation to be effective. Cell survival is inversely proportional to the electrical energy dissipated (joules) in the medium. Electroporation is a convenient and effective means of introducing transforming DNA into T. thermophila.     

A specific uncoupler-binding protein in Tetrahymena pyriformis and Paracoccus denitrificans

The uncoupler of mitochondrial oxidative phosphorylation, 2-nitro-4-azido-carbonylcyanide phenylhydrazone (N₃CCP) which is capable of photoaffinity labeling has been used to examine the effect of uncouplers on the energy conserving membranes of Paracoccus denitrificans and Tetrahymena pyriformis. The N₃CCP uncouples respiration in P. denitrificans and T. pyriformis cells with $\text{U}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values of 1.05 μM and 0.24 μM, respectively. Binding studies show the presence of 0.65 ± 0.05 high affinity sites per cytochrome a with a K_d of 0.5 ± 0.1 μM in P. denitrificans membranes and 1.4 ± 0.2 sites per cytochrome a₂ with a K_d of 0.4 ± 0.1 μM in T. pyriformis membranes. Irradiation of [³H]-N₃CCP bound to the membranes leads to a covalent linking of the radioactive uncoupler to a peptide of 10–15 kdaltons as analyzed by SDS-polyacrylamide gel electrophoresis. It is concluded that these two microbial systems contain a specific high affinity uncoupler binding site very similar to that of mammalian mitochondria (Katre, N.V. and Wilson, D.F. (1978) Arch. Biochem. Biophys. 191, 647–656).     

梨形四膜虫衰老过程中DNA含量的变化

应用一种新型的细胞核内DNA含量测定方法──图像分析法,测定真核细胞梨形四膜虫衰老过程中DNA含量的变化.根据Beer-Lambert定律,以细胞核在不同生长期内的积分光密度的水平表示核内DNA含量的变化.该方法具有测量速度快,重复性好,操作简单,结果可靠等优点.实验结果表明:四膜虫在进入对数生长期时,DNA含量逐渐达到高峰,随着细胞逐渐老化,细胞分裂次数及核内DNA含量逐步减少.     

Analysis of nuclei from exponentially growing and conjugated Tetrahymena thermophila using the flow microfluorimeter

Isolated nuclei of Tetrahymena thermophila from both exponentially growing cultures and from cells following conjugation have been analysed using a flow microfluorimeter. The macronuclei from a culture in exponential growth display a single broad distribution of DNA contents, without bimodal character. The micronuclei are virtually all in G2 phase (4C). The mean of the macronuclear DNA distribution is about 12.4 times the micronuclear mean (50C). When cells are starved in preparation for conjugation, the macronuclei DNA content is decreased about 30%, but the distribution remains similar to that of nuclei from a culture in exponential growth. Following conjugation, the macronuclear anlagen develop through a set of relatively synchronous endoreplications. At 12 h after the initiation of conjugation the anlagen are at a 4C stage and at 18 h they are virtually all at a 8C stage. If the culture is refed, anlagen development progresses to a 16C and 32C, but the synchrony is poorly conserved. Cells that are not refed are arrested at the 8C stage and only a fraction of the population ever become mature macronuclei. In general we do not observe distinct peaks of anlagen with DNA contents in excess of 32C. The amitotic division of macronuclei may obscure any endoreplications producing anlagen stages with higher DNA content.     

Proteolytic processing of micronuclear H3 and histone phosphorylation during conjugation in Tetrahymena thermophila

During vegetative growth, micronuclei of the ciliated protozoan Tetrahymena thermophila contain two electrophoretically distinct forms of H3, H3S and H3F [4, 5]. Of these two forms, H3F is unique to micronuclear chromatin and is derived from H3S by a physiologically regulated proteolytic processing event [5]. While the function of this processing event is not clear, several lines of evidence [2, 5] suggest that it may be related to chromatin condensation during mitosis. In this report pulse-chase experiments have been used to study the processing of H3S into H3F during the sexual phase of the life cycle, conjugation. Our results demonstrate that even though micronuclei divide mitotically (and meiotically) several times during the mating process, processing of H3S into H3F does not occur. Failure of H3S to be converted into H3F during these divisions causes a significant increase in the amount of H3S (relative to H3F) as conjugation proceeds. By 10 h of conjugation, essentially all of the micronuclear H3 is in the form of H3S (also see [3]). As long as mating cells are maintained under starvation conditions, processing of H3S into H3F does not occur. However, if exconjugants are returned to food and allowed to proceed through the first true cell division following exconjugation, processing of H3S into H3F occurs. Thus, the return of the processing of H3(3) into H3F following conjugation seems to be tightly coupled to a division which is part of a cell division cycle (as appears to be the case with vegetatively growing cells). The relevancy of these results to the differentiation of new macro- and micronuclei is discussed. H3F is specifically phosphorylated in growing cells, and it has been suggested that this phosphorylation event may be related to chromatin condensation during mitosis [2]. Since in mating cells H3S becomes the more predominant form of H3, the pattern of histone phosphorylation was examined during stages of conjugation where micronuclei are active in mitotic division (6-7 h). While a low level of phosphate label is observed over H3S in mating cells, more phosphate label is associated with the small amount of H3F which remains in micronuclei at this stage of conjugation. We also observe significant amounts of phosphate label associated with micronuclear H2A, H2B, and H4 and each of the micronuclear H1-like molecules, alpha, beta and gamma.     

The dynein heavy chain gene family in Tetrahymena thermophila

The dynein ATPases are a family of motor enzymes that drive microtubule sliding in cilia and flagella and contribute to microtubule-based transport inside cells. The multi-dynein hypothesis makes two predictions: 1) Axonemes contain multiple dynein heavy chain (DHC) isoforms, each encoded by a different gene; 2) Each isoform performs a specific role in ciliary beating. We used PCR-based techniques to clone thirteen different DHC sequences from Tetrahymena genomic DNA. All thirteen genes appeared to be expressed in growing cells. Comparisons of the deduced amino acid sequences of the thirteen DHCs with other known DHCs suggested that we have cloned three outer arm DHCs, two cytoplasmic DHCs, and eight inner arm DHCs.     

Breeding performance of Tetrahymena thermophila following storage for 5 to 6 years in liquid nitrogen

Although the ciliated protozoan, T. thermophila, approaches immortality while growing vegetatively, most stocks sooner or later lose the ability to produce viable progeny after conjugation. Liquid nitrogen storage eliminates, or drastically slows, this aging process.Multiple stocks of 11 inbred strains have been thawed after 1,2, and 5 to 6 years in storage and mated to a tester strain or among themselves. Significantly more viable progeny resulted from crosses of frozen-thawed cultures than from crosses of unfrozen controls. The performance of the inbred strains varied as did that of replicate samples from rapidly aging strains. Crosses made with samples in which relatively few cells had survived storage and thawing varied more than did those made with samples containing many survivors.These results (a) show that good breeding strains retain this ability during storage and (b) support the hypothesis that the mechanism of aging in Tetrahymena results from the accumulation of random defects rather than an intrinsic program. It appears, however, that no results from the present experiments provide any new insight into the mechanism of aging.     

Wild-type and food-vacuole-less Tetrahymena thermophila : Model for iron uptake and utilization in animal cells

Wild-type Tetrahymena thermophila and a conditional lethal mutant defective in food-vacuole formation constitute a useful model system for studies of iron uptake and utilization in animal cells.