Kinetics of the DFPase activity in Tetrahymena thermophila

Crude homogenates of the ciliate protozoon, Tetrahymena thermophila, can hydrolyze the potent acetylcholinesterase inhibitors O,O-diisopropylphosphorofluoridate (DFP) and O-1,2,2-trimethylpropylmethylphosphonofluoride (soman). Characterization of the enzymatic activity of the homogenate has been performed. The DFPase operates over a pH range of 4 to 10 and an ionic range of 0-500 mM NaCl. Rate of reaction increases three- to four-fold from 25 degrees C to 40 degrees C and is still present at 55 degrees C. These results indicate that the enzymatic activity operates over a broad range of environmental conditions, making it an attractive material for use in the detoxification and detection of organofluorophosphates. DFPases may be important in the metabolism of naturally occurring organophosphates.     

An Organofluorophosphate-Hydrolyzing Activity in Tetrahymena thermophila1

An enzymatic activity that hydrolyzes O,O-diisopropylphosphofluoridate (DFP) and O-1,2,2–trimethylpropylmethyl-phosphonofluoridate (Soman) was discovered in the ciliate protozoan Tetrahymena thermophila. The enzymatic activity classifies the protein as Mazur-type similar to that found in hog kidney and Escherichia coli. The rate of hydrolysis of Soman by the Tetrahymena-extract is the highest, on a per gram of extract basis, of any eucaryote. The molecular weight is approximately 75,400 as determined by Sephacryl column chromatography. A maximum fifteen-fold purification has been achieved. Potential exists for the detoxification and one-step detection of common organofluorophosphate pollutants. Additionally, Tetrahymena should prove an easier subject for manipulation than mammalian or squid sources. Protozoa may be a potentially important source of detoxification and degradation enzymes for other environmental contaminants.     

Glutathione in Tetrahymena thermophila

In Tetrahymena , glutathione is synthesized from the same precursors as it is in higher animals and is present in similar intracellular concentrations. The intracellular thiol-disulfide ratio is also identical to that of mammalian tissues, due to the activity of glutathione reductase. The intracellular GSH-level was found to be dependent on the sulfur-containing amino acids in the chemically defined medium.     

Glutathione in Tetrahymena thermophila

In Tetrahymena, glutathione is synthesized from the same precursors as it is in higher animals and is present in similar intracellular concentrations. The intracellular thiol-disulfide ratio is also identical to that of mammalian tissues, due to the activity of glutathione reductase. The intracellular GSH-level was found to be dependent on the sulfur-containing amino acids in the chemically defined medium.     

Constitutive and Induced Excretion of Polypeptides in Tetrahymena thermophila1

Polypeptides normally excreted to growth or starvation media were revealed using O'Farrell gel-electrophoresis and silver-staining. The major polypeptides detected in conditioned media were either constitutive, growth specific, starvation specific, or high-Tris specific. The majority of the excreted polypeptides could be released from the cells by a hypo-osomotic shock, possibly resulting in membrane leakage, but also mucocyst and/or plasma membrane-associated polypeptides were detected in the conditioned media.     

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.     

Effect of Biogenic Amines on Phagocytosis in Tetrahymena thermophila1

Stimulation of phagocytosis by serotonin and catecholamincs in Tetrahymena grown in proteose-peptone medium proved to be concentration dependent, the optimal concentrations being ∼0.1 to 1.0 μM. The serotonergic antagonists, spiperone, and metergoline, also stimulated the process, whereas the β- and α-adrenergic antagonists, propranolol, alprenolol, and ergocryptine, had no effect or inhibited phagocytosis. A wide variety of derivatives of the biogenic amines had no effect on phagocytosis, demonstrating the specificity of recognition mechanism for neurohormones in Tetrahymena. Such hormones act by at least two independent mechanisms, one for adrenergic agonists, another for dopamine. Presumably, recognition mechanisms for hormones in protozoa resemble in some respects those in multicellular organisms, therefore bespeaking a common origin.     

RAN1基因过表达抑制嗜热四膜虫大核无丝分裂

Ran GTPase通过RanGTP/RanGDP循环的形式,参与调控多种细胞增殖方式：包括有丝分裂和减数分裂.敲减RAN1基因可导致嗜热四膜虫大核内微管组装紊乱,从而抑制大核无丝分裂.为进一步分析Ran1在无丝分裂中的功能,本研究将野生型Ran1以及模拟GTP（Ran1Q70L）和GDP（Ran1T25N）锁定形式的Ran1突变体在嗜热四膜虫中过量表达,均导致四膜虫细胞增殖速率下降,并引起大核无丝分裂异常,且这种核异常细胞比率与Ran1过表达量呈正相关.免疫荧光定位结果显示,过表达的HA-Ran1在整个细胞中弥散分布,破坏了正常的Ran1分布形式;而过表达的HA-Ran1Q70L明显集中在大核核膜和胞质中,HA-Ran1T25N则主要定位在大核和小核内,分别与Ran1GTP/Ran1GDP循环的辅助调节因子定位模式一致.以上结果表明,过表达Ran1及其突变体可能影响嗜热四膜虫细胞中正常的Ran1GTP/Ran1GDP循环,进而导致大核无 丝分裂异常.     

S-Adenosylhomocysteine Hydrolase Deficiency in Cysteine Auxotrophs of Tetrahymena thermophila1

The biochemical lesion in two cysteine auxotrophs of Tetrahymena thermophila has been established as a defect in S-adenosylhomocysteine hydrolase, an enzyme of the transsulfuration pathway. As a result, these mutants require cysteine (or cystathionine or homocysteine) for growth in a denned medium. Cell-free extracts of the mutants contained < 5% of the level of the enzyme seen in the wild type. One of the mutant strains accumulated intracellular levels of S-adenosylhomocysteine as high as 1380 üM, a level 200 times normal. When both mutant strains were maintained in defined medium without cysteine, growth occurred after a long lag; this phenomenon was termed “adaptation.” Adaptation was a) reversed by passage through rich medium, b) was not a recovery of S-adenosylhomocysteine hydrolase, and c) was probably linked to induction of an alternate pathway for cysteine biosynthesis, involving a lysosomal S-adenosylhomocysteine nucleosidase activity.     

Biogenic Amines Stimulate Regeneration of Cilia in Tetrahymena thermophila1

ABSTRACT Serotonin and catecholamines affect the regeneration of cilia in Tetrahymena thermophila in a dose-dependent manner: micromolar concentrations are stimulatory, whereas millimolar concentrations have little or no effect. This conclusion is based on motility measurements in regenerating cells and on ciliary counts in scanning electron micrographs. In addition, the recognition mechanism for each hormone appears to be specific and independent. Our results suggest an evolutionary link with hormonal mechanisms in multicellular eukaryotes.     

抗沉默因子Asf1调控嗜热四膜虫细胞核的稳定性

组蛋白H3/H4的分子伴侣Asf1(anti-silencing factor 1),参与依赖DNA复制及不依赖DNA复制的核小体装配,同时参与转录调控、基因沉默以及DNA损伤修复等过程. 在不同生物中,Asf1具有功能的保守性和多样性．嗜热四膜虫ASF1（TTHERM_00442300）基因编码的蛋白质含有保守的N端结构域和酸性的C端结构域．N端结构域同源序列进化树分析表明,Asf1进化与物种进化一致．实时荧光定量PCR表明,ASF1在四膜虫营养生长、饥饿及有性生殖时期均有表达,且在有性生殖4～6 h转录水平达到最高．免疫荧光定位分析表明,HA-Asf1在营养生长时期以及有性生长时期定位于功能大核和小核中,而在凋亡的大核中信号消失．过表达ASF1导致大核及小核变大,抑制细胞增殖．敲减ASF1后会导致大核形态异常,小核缺失．结果表明,ASF1表达对细胞核的形态和结构维持发挥重要的调控作用．     

Small RNAs of Tetrahymena thermophila

Highly purified nuclear and cytoplasmic RNAs were obtained from Tetrahymena thermophila BVII containing only a minimal amount of cross-contamination. In the nuclear RNA fraction we have detected at least 6 distinct snRNAs. Some of the RNA species showed microheterogeneity. SnRNAs of Tetrahymena thermophila are very similar to rat snRNAs, as far as length is concerned. Our cytoplasmic small RNA fraction contained two RNAs, 7S and T7, reported recently (18) as nuclear, particularly nucleolar RNAs. Moreover, we could detect only one cytoplasmic small RNA species Tc1, Tc2 was not observed.Neither the nuclear nor the cytoplasmic small RNA species are degradation products of ribosomal RNA as was shown by Northern blotting and following hybridization with pGY17 containing the entire transcribed region of the ribosomal DNA of Tetrahymena thermophila.     

Isolation and Properties of Macronuclei from Tetrahymena thermophila1

A simple and efficient method is described for the isolation of macronuclei from Tetrahymena thermophila (7B). The steps involved are deciliation and removal of the mucocysts’ contents by dibucaine treatment, digitonin mediated lysis, differential centrifugations, and finally isopyenic sucrose density gradient centrifugation. Judging from the distribution of marker enzymes and electron microscopy, the macronuclei obtained were free of cytoplasmic and paniculate contamination and were highly active in endogenous RNA-synthesis (1.5 pmol UTP incorporation/ng DNA min at 30°C). The ratio of protein: RNA: DNA was 2.0:0.33:1.0 (weight) and each macronucleus contained an average of 17 pg DNA. The average yield of isolation was 50%.     

Biochemical Approaches to Mating Type Expression in Tetrahymena thermophila1

Two-dimensional gel electrophoresis was used to identify the patterns of protein synthesis during initiation, and the patterns of membrane protein expression following initiation, in all of the mating types of the Tetrahymena thermophila B family. In addition, one-dimensional analysis was used to survey ¹²⁵I-Concanavalin A-binding proteins. Although a large number of proteins was identified by each technique, no variation among the mating types was observed.     

DYF-1 Is Required for Assembly of the Axoneme in Tetrahymena thermophila

In most cilia, the axoneme can be subdivided into three segments: proximal (the transition zone), middle (with outer doublet microtubules), and distal (with singlet extensions of outer doublet microtubules). How the functionally distinct segments of the axoneme are assembled and maintained is not well understood. DYF-1 is a highly conserved ciliary protein containing tetratricopeptide repeats. In Caenorhabditis elegans, DYF-1 is specifically needed for assembly of the distal segment (G. Ou, O. E. Blacque, J. J. Snow, M. R. Leroux, and J. M. Scholey. Nature. 436:583-587, 2005). We show that Tetrahymena cells lacking an ortholog of DYF-1, Dyf1p, can assemble only extremely short axoneme remnants that have structural defects of diverse natures, including the absence of central pair and outer doublet microtubules and incomplete or absent B tubules on the outer microtubules. Thus, in Tetrahymena, DYF-1 is needed for either assembly or stability of the entire axoneme. Our observations support the conserved function for DYF-1 in axoneme assembly or stability but also show that the consequences of loss of DYF-1 for axoneme segments are organism specific.Cilia are microtubule-rich cellular extensions that arise from basal bodies near the surfaces of most eukaryotic cell types. Defective cilia cause a wide variety of diseases, including polycystic kidney disease, primary ciliary dyskinesia, and retinal degeneration (3). A typical motile cilium has a microtubule-based framework, the axoneme, which contains nine outer (mostly doublet) microtubules and two central (singlet) microtubules. In most cilia, the axoneme can be subdivided into three segments: proximal (transition zone), middle (containing outer doublet microtubules), and distal (containing singlet extensions of peripheral microtubules). The outer doublet microtubules of the middle segment have a complete tubule A made of 13 protofilaments and an incomplete tubule B made of 11 protofilaments that is fused to the wall of the A tubule (36, 57). The outer microtubules in the distal segment lack the B tubule (32, 49). The distal segment also lacks dynein arms and radial spokes, and its microtubules are terminated by caps that are associated with the plasma membranes at the tips of cilia (11, 50). The distal segments are characterized by a high level of microtubule turnover, which could play a role in the regulation of the length of cilia (31).The mechanisms that establish the segmental subdivision of the axoneme are not well understood. Studies of Caenorhabditis elegans indicate that the distal segment is assembled using a mechanism that differs from the one utilized in the middle and proximal segments (54). In most cell types, ciliogenesis is dependent on the intraflagellar transport (IFT) pathway, a bidirectional motility of protein aggregates, known as IFT particles, that occurs along outer microtubules (10, 28, 29, 42). IFT particles are believed to provide platforms for transport of axonemal precursors (23, 44). The anterograde component of IFT that delivers cargo from the cell body to the tips of cilia is carried out by kinesin-2 motors (28, 63), whereas the cytoplasmic dynein DHC1b is responsible for the retrograde IFT (41, 43, 53). Importantly, in the well-studied amphid cilia of C. elegans, two distinct kinesin-2 complexes are involved in the anterograde IFT and differ in movement velocity: the “slow” heterotrimeric kinesin-II and the “fast” homodimeric OSM-3 kinesin (54). While kinesin-II and OSM-3 work redundantly to assemble the middle segment, OSM-3 alone functions in the assembly of the distal segment (39, 56).In C. elegans, DYF-1 is specifically required for assembly of the distal segment (39). In the DYF-1 mutant, the rate of IFT in the remaining middle segment is reduced to the level of the slow kinesin-II, suggesting that the Osm3 complex is nonfunctional and that kinesin-II functions alone in the middle segment. Thus, DYF-1 could either activate OSM-3 kinesin or dock OSM-3 to IFT particles (14, 39).However, a recent study of zebrafish has led to a different model for DYF-1 function. Zebrafish embryos that are homozygous for a loss of function of fleer, an ortholog of DYF-1, have shortened olfactory and pronephric cilia and ultrastructural defects in the axonemes. In the middle segment, the fleer axonemes have B tubules that are disconnected from the A tubule, indicating that DYF-1 functions in the middle segment and could play a role in the stability of doublet microtubules (40). Earlier, a similar mutant phenotype was reported in Tetrahymena for a mutation in the C-terminal tail domain of β-tubulin, at the glutamic acid residues that are used by posttranslational polymodifications (glycylation and glutamylation) (47). Glycylation (46) and glutamylation (12) are conserved polymeric posttranslational modifications that affect tubulin and are highly enriched on microtubules of axonemes and centrioles (reviewed in reference 20). Other studies have indicated that tubulin glutamylation contributes to the assembly and stability of axonemes and centrioles (4, 8). The fleer mutant zebrafish cilia have reduced levels of glutamylated tubulin (40). Pathak and colleagues proposed that the primary role of DYF-1/fleer is to serve as an IFT cargo adapter for a tubulin glutamic acid ligase (25) and that the effects of lack of function of DYF-1/fleer could be caused by deficiency in tubulin glutamylation in the axoneme (40). As an alternative hypothesis, the same authors proposed that DYF-1 is a structural component that stabilizes the doublet microtubules in the axoneme (40).Here, we evaluate the significance of a DYF-1 ortholog, Dyf1p, in Tetrahymena thermophila. Unexpectedly, we found that Tetrahymena cells lacking Dyf1p either fail to assemble an axoneme or can assemble an axoneme remnant. While our observations revealed major differences in the significance of DYF-1 for segmental differentiation in diverse models, it is clear that DYF-1 is a conserved and critical component that is required for assembly of the axoneme.     

Oral Assembly in Left-Handed Tetrahymena thermophila

We have investigated oral development in a non-genically derived left-handed (LH) form of Tetrahymena thermophila , in which the large-scale asymmetry of arrangement of cortical structures is reversed whereas the local asymmetry of ciliary architecture remains normal. Approximately 1/2 of the oral apparatuses (OAs) of LH cells develop in the form of superficial mirror-images of OAs of RH cells. In most of these OAs, membranelles are assembled from the cells'anterior to posterior. Nonetheless, the posterior ends of these membranelles undergo the basal body displacements that lead to a "sculptured" appearance, so that the membranelles of LH OAs become organized as rotational permutations of membranelles of normal RH OAs. Many of these membranelles re-orient to a normal orientation near the end of oral development. Membranelles and undulating membranes (UMs) may develop independently of each other, and formation of postciliary microtubules of UMs is separate from that of ribbed wall microtubules. In some cases, the entire OA develops and remains as a 180° rotational permutation of the normal, resembling the inverted OAs of mirror-image doublets and LH cells of Glaucoma scintillans described by Suhama [36, 37]. We present a model (Fig. 37) for these complex developmental outcomes. These developmental patterns resemble those described previously and less completely for "secondary" OAs of cells with mirror-image global patterns, including janus cells. The present study demonstrates that such alterations in oral development are not a direct outcome of genotypic changes.     

嗜热四膜虫减数分裂研究进展

减数分裂是真核生物有性生殖过程的关键步骤,染色体的行为变化贯穿整个减数分裂的过程。近些年来,借助先进的分子生物学技术和细胞学实验手段,通过对突变细胞株的筛选和评价,单细胞真核模式生物原生动物嗜热四膜虫(Tetrahymena thermophila)减数分裂方面的研究取得了长足的进展。本文主要介绍嗜热四膜虫减数分裂的过程,以及在此过程中伴随染色体行为变化的相关基因的功能,从而为进一步探讨嗜热四膜虫减数分裂的分子机制提供有效信息。     

Putrescine biosynthesis in Tetrahymena thermophila.

The putrescine-biosynthesis pathway in Tetrahymena thermophila was delineated by studying crude extracts prepared from exponentially growing cultures. A pyridoxal phosphate-stimulated ornithine decarboxylase activity competitively inhibited by putrescine was detected. CO2 was also liberated from L-arginine, but analyses by t.l.c. and enzyme studies suggested that the activity was not due to arginine decarboxylase, nor could enzyme activities converting agmatine into putrescine be detected. We conclude that the decarboxylation of L-ornithine is probably the only major route for putrescine biosynthesis in this organism during exponential growth.     

Cilia-Mediated Oriented Chemokinesis in Tetrahymena thermophila

The role of the cilia in the locomotion (“gliding”) of Tetrahymena thermophila in a semi-solid medium has been studied when cells were migrating in gradients of attractant. Video recordings and computer-aided motion analysis of migrating cells and their ciliary activity show that Tetrahymena thermophila migrate by swimming forward in semi-solid methyl cellulose, using their cilia. Ciliary reversals occur at certain intervals and cause a termination (“stop”) of cellular migration. Cells with reversed cilia resume forward migration when normal ciliary beating resumes. In gradients of attractants, cells migrating towards the attractant suppress ciliary reversals, which leads to longer runs between stops than in control cells. Cells migrating away from the attractant have a higher frequency of ciliary reversals than the control cells resulting in shorter runs. Stimulated cells adapt to a particular ambient concentration of attractant several times during migration in the gradient. Adaptation is followed by de-adaptation, which occurs during the “stop”. In the presence of cycloheximide, a strong inhibitor of chemoattraction, the attractant-induced suppression of ciliary reversal is abolished (cells become desensitized to the attractant). It is concluded that Tetrahymena has a short-term memory during adaptation. This is important for the efficiency of migration towards an attractant.