Fine Structure, Reconstruction and Possible Functions of Components of the Cortex of Tetrahymena pyriformis

SYNOPSIS. Improved methods of preparing cells for electron microscopic study have permitted a more complete examination of the tubular and fibrous elements already reported in the cortex of Tetrahymena pyriformis. Three of these structures, a kinetodesmal fiber, a band of postciliary microtubules and a band of transverse microtubules, are all associated at one of their ends with the proximal end of a basal body-cilium complex. From this position they radiate out as tho from one corner of a tetrahedron, each passing separately to one of the other 3 corners. Available evidence is presented and discussed for a structural function for these elements. A firm bonding of these fibers and bands to the basal body at one of their ends and to the amorphous material under the pellicle at their other ends is thought to provide for this support. Connecting the proximal ends of the left side of the basal bodies of a kinety is another, previously undescribed, set of tubules. Their diameter, 24 mμ, and cross sectional structure are similar to those of the other microtubules. However, their more sinuous longitudinal appearance, small number of tubules per kinety and different reaction to fixatives suggest a different function. Because of their location it is proposed that they may be a communication line between basal body-cilium complexes of a kinety. A 3-dimensional drawing shows the positioning of the above structures in the cortex. Bodies with an internal tubular structure appear anterior to the proximal end of some basal bodies. They are referred to as probasal bodies due to their resemblance to procentrioles; they may be immature basal bodies. Smooth-membraned cisternae which have bristle-coated pits and bear a resemblance to the outer pellicular membrane appear in the cytoplasm. Their origin is discussed.     

Tetrahymena thermophila: analysis of phospholipids and phosphonolipids by high-field 1H-NMR

The phospholipids of control and lipid-modified Tetrahymena thermophila were identified and quantified, using 1-D and 2-D COSY proton NMR spectroscopy on intact lipids, before and after HPLC separation. The results are comparable to those obtained using classical lipid analytical techniques. The results indicate that the study of enzyme pathways and other metabolic processes involving phospholipids in Tetrahymena and related protozoa can be carried out using proton NMR spectroscopy as the investigating technique.     

Membrane Protein Differences Correlated with the Development of Mating Competence in Tetrahymena thermophila1

Initiation is the contact-independent phase of sexual conjugation which occurs when mature cells of Tetrahymena thermophila are shifted from growth medium to a low-salt starvation buffer. Immaturity, like high-salt starvation, restricts the ability of cells to conjugate; immature cells do not conjugate in either low- or high-salt buffers. Comparisons between sexually mature cells starved in initiation-restrictive and initiation-permissive buffers, and between immature and mature cells starved in an initiation-permissive buffer permitted the analysis of membrane protein expression correlated with mating competence. No polypeptides identified by lactoperoxidase-catalyzed iodination were found to be specific to mating-competent cells; however, several polypeptides not present in initiated cells were found to be common to the cell surfaces of immature and non-initiated cells which suggests that (1) initiation involves the removal of specific proteins from the cell surface, and (2) immaturity may be due to an inability to initiate.     

Purification and Partial Characterization of the H Immobilization Antigens of Tetrahymena thermophila1

ABSTRACT. The H immobilization antigens specified by the SerH locus of Tetrahymena thermophila have been purified by a procedure utilizing acid fractionation, ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. Purified antigen migrates as a single band on SDS-PAGE and IEF. Molecular weights of the four allelic H antigens range from 44,000 to 52,000, and isoelectric points range from 4.1 to 4.5. No carbohydrate was detected.     

上海四膜虫和两株嗜热四膜虫的rRNA基因ITS-1序列及分子系统关系

测定并比较了自接型的上海四膜虫（Tetrahymena shanghaienisis)和两株接合型的嗜热四膜虫（T.thermopddhilaⅡ和T.thermophilaⅥ）的ITS-1序列,以多态嗽叭虫（Stentor polymorphrus)为外来群,利用最大简约法和邻接法构建了它们的系统发育树。分析指出：三者中,T.shanghaienisis较早地从祖先种中分化出来;自接型可能是一种较接合型原始的生殖方式。     

Development of the Ciliary Pattern of the Oral Apparatus of Tetrahymena thermophila1

ABSTRACT. The sequence of formation and ciliation of basal bodies and the subsequent organization of compound ciliary structures of the oral apparatus of Tetrahymena thermophila was reanalyzed with the aid of scanning electron microscopy of cells in which the epiplasmic layer was exposed, as well as by light microscopy of protargol-impregnated specimens. This combination of methods allowed the delineation of numerous steps in the patterning of the oral ciliature, some of which have received little or no previous attention. Highlights include: the initial formation of “strings” of nonciliated new basal bodies in juxtaposition to relatively few basal bodies of the stomatogenic kinety; generation of basal body pairs, roughly oriented along the anteroposterior axis of the cell, that later align side-by-side to assemble promembranelles; condensation and reorientation of promembranelles simultaneous with addition of a third row of basal bodies anterior to the original two rows; production of a very short fourth row of basal bodies at the anterior right end of each developing membranelle; generation of the outer basal body row of the undulating membrane (UM) after alignment of the inner row, with transient ciliation of the inner row preceding permanent ciliation of the outer row; limited basal body resorption at the ends of membranelles; and sculpturing of the right ends of membranelles by a movement of basal bodies associated with formation of the ribbed wall adjacent to the UM. In the old anterior oral apparatus a repetition of the processes of generation of a new outer UM row and sculpturing of right ends of membranelles takes place in synchrony with the corresponding events in the oral primordium, following prior shedding of the old outer UM row and loss of the sculptured pattern in association with temporary regression of the ribbed wall micro-tubules. Oral development is complex, with different processes involved in the assembly of the membranelles and the UM, and with a sequence of distinct events involved in the generation of each of these structures. Speaking comparatively, membranelle development follows the same pathway in many, perhaps all, ciliates in which these structures or their homologues develop from a common stomatogenic field.     

嗜热四膜虫染色体浓缩调节因子(RCC1)的定位及功能分析

真核细胞中染色体浓缩调节因子（regulator of chromosome condensation 1, RCC1）是 RanGTPase 唯一的鸟嘌呤核苷酸交换因子. 染色质结合的RCC1和RanGTPase相互作用,催化细胞核内RanGDP向RanGTP的转化,进而调控了核质间的定向运送、有丝分裂期纺锤体的组装以及核膜的形成. 本实验从原生生物嗜热四膜虫大核基因组中鉴定了1个新的RCC1（TTHERM_00530380）基因. 该基因全长2 541 bp,包含2个内含子序列,开放阅读框为2 181 bp,编码726个氨基酸. 实时荧光定量PCR表明,RCC1在四膜虫营养生长、饥饿以及有性生殖时期都有表达,且在有性生殖转录水平达到最高. 免疫荧光定位分析表明, HA RCC1在营养生长和饥饿时期,定位于大核和小核中|在有性生殖时期,定位于亲本大核、减数分裂的小核、新生成的大核和凋亡的大核中. 过表达RCC1导致大核的无丝分裂异常, 细胞增殖变慢,最终产生无大核的后代细胞. 敲减RCC1导致了多小核的产生. 结果表明,RCC1参与调控了四膜虫细胞核的分裂, RCC1的正常表达对核分裂以及细胞增殖起到重要的调控作用.     

Overexpression and purification of recombinant eRF1 proteins of rabbit and Tetrahymena thermophila

The polypeptide release factor (eRF1) gene was cloned from rabbit and its overexpression and purification system was established in parallel with that of the eRF1 gene of Tetrahymena thermophila that has been cloned recently in this laboratory. The rabbit eRF1 (Ra-eRF1) is composed of 437 amino acids and is completely identical to human eRF1 though 3% distinct in the nucleotide sequence. This is in sharp contrast to Tetrahymena eRF1 (Tt-eRF1) that is only 57% identical to human eRF1. The recombinant Ra-eRF1 was marked with a histidine tag, overexpressed, and purified to homogeneity by two-step chromatography using Ni-NTA-agarose and Mono Q columns. In contrast to Ra-eRF1, Tt-eRF1 formed aggregates upon overexpression in Escherichia coli, hence it was purified under denaturing conditions, and used to raise rabbit antibody. The resulting anti-Tt-eRF1 antibody proved useful for examining conditions for soluble Tt-eRF1 in test cells. Finally, a soluble Tt-eRF1 fraction was purified from Saccharomyces cerevisiae transformed with the Tt-eRF1 expression plasmid by three steps of affinity and anion exchange chromatography. The cloned Ra-eRF1 gene complemented a temperature-sensitive allele in the eRF1 gene, sup45 (ts), of S. cerevisiae, though the complementation activity was significantly impaired by the histidine tag, whereas Tt-eRF1 failed to complement the sup45 (ts) allele.     

Ecological Genetics of Tetrahymena thermophila: Mating Types, i-Antigens, Multiple Alleles and Epistasis

Until recently, Tetrahymena thermophila has rarely been isolated from nature. With improved sampling procedures, T. thermophila has been found in ponds in many northeastern states. The availability of resident populations makes possible both population and ecological genetic studies. All seven known mating types have been recovered; no eighth mating type has been found. Crosses among whole-genome homozygotes derived from Pennsylvania isolates reveal a spectrum genotypes with mating type alleles resembling traditional A (IV- and VII-) and B(I-) categories. The genotypes differ significantly with respect to mating type frequency, both among themselves and from previously described genotypes. One A-category genotype appears to lack mating type II, while one A-category and all B-category genotypes have low frequencies of mating type III, thus accounting for the low frequency of III in the pond. The low frequency of III in all five B-category genotypes examined suggests that the founding allele in this region was low for III. These and other differences are discussed both in terms of mating type frequencies in the pond and in terms of the possible molecular structure of mat alleles. By contrast, numerous variants of the cell surface immobilization antigen are found in addition to the previously described i-antigens. Variants of the known SerH alleles include those with restriction fragment length polymorphisms and temperature sensitivity as well as alleles with new antigenic specificity. Multiple alleles are present in single ponds. Genes exhibiting serially dominant epistasis over SerH genes also are found. In two instances (K and C), families of antigenically similar polypeptides are expressed in place of H i-antigen. Molecular weight differences suggest that these paralogous i-antigen genes evolve by gene duplication and unequal crossing over within central repeats. The existence of complex patterns of epistasis together with seasonal changes in i-ag frequencies suggest that i-ag play an important, but as yet unknown, ecological role related to the occurrence of frequent conjugation.     

Alternate Junctions and Microheterogeneity of Tlr1, a Developmentally Regulated DNA Rearrangement in Tetrahymena thermophila

ABSTRACT. A large number of developmentally regulated DNA rearrangements occur during the development of the macronucleus in Tetrahymena thermophila , Tlr1 is a deletion element which has large inverted repeats near the rearrangement junctions and deletes more than 13 kbp of internal DNA. Previous analysis of caryonidal lines revealed alternate left junctions for the Tlr1 rearrangement in B strain cells. We show here that C2 strain Tetrahymena also use alternate rearrangement junctions. We have mapped and sequenced two additional rearrangement variants and find that both the left and right can vary over a range of approximately 200 bp. We also demonstrate the presence of sequence microheterogeneity in the most commonly found Tlr1 rearrangement product.     

嗜热四膜虫胱硫醚γ-裂解酶Cgl1的表达、定位及功能分析

含硫氨基酸在不同的生物体中具有重要调节功能,转硫途径相关酶促进半胱氨酸的生成和硫化氢产生。本研究从嗜热四膜虫中鉴定一种胱硫醚γ-裂解酶（cystathionine γ-lyase 1,CGL1,TTHERM_00052400）基因。CGL1在营养生长期高水平表达,而在饥饿阶段和有性生殖期,维持在较低的表达水平。通过密码子优化,人工合成CGL1基因,构建重组表达质粒pGEX-CGL1,转化大肠杆菌BL21(DE3)。E.coli/pGEX-CGL1表达重组蛋白质GST-Cgl1,并通过亲和层析获得纯化。GST-Cgl1裂解胱硫醚产生半胱氨酸,也具有裂解半胱氨酸和同型半胱氨酸产生H2S的活性。进一步构建重组质粒pNEO4-3HA-CGL1和pSMC1hpNEO-CGL1,转化四膜虫细胞,获得带有HA标签和干扰CGL1的突变体细胞株。免疫荧光定位表明,HA-Cgl1生长期定位在亲本大核,饥饿期定位在细胞质,有性生殖前期定位在亲本大核,而在后期定位在胞质中。CGL1干扰的突变体细胞株在有性生殖过程中不能形成合子核,发育中的小核异常降解,产生仅有大核的异常单细胞。结果表明,嗜热四膜虫含有进化中保守的胱硫醚γ-裂解酶Cgl1。Cgl1具有产生和裂解半胱氨酸的活性。Cgl1定位在细胞质和细胞核中,参与了有性生殖过程细胞核的发育。     

Screening for and characterization of phospholipase A1 hypersecretory mutants of Tetrahymena thermophila

We have described a procedure for the isolation of mutants of Tetrahymena thermophila with hyperscretion of phospholipase A₁ (PLA₁). Using random chemical mutagenesis, uniparental cytogamy, genetic crossing and a new, fast and effective screening procedure, four PLA₁-hypersecretory mutants were isolated. The screening procedure is based on the formation of a halo appearing around cylindrical holes in a lecithin-containing agar plate filled with cell-free supernatants. About 3,940 clones were tested with this procedure in primary screening for hypersecretory features, of which 60 putative hypersecretory mutants were isolated, subcloned and tested in a secondary screening. Of these, four selected mutants showed 1.8–2.2 more PLA₁ activity in the cell-free supernatants compared to the wild-type strain CU 438.1. Hypersecretion was only observable for PLA₁; no increased activity for two other lysosomal enzymes could be detected. These hypersecretory mutants of T. thermophila can be very useful for increasing the yield of PLA₁ in fermentation processes. This is particularly relevant because, in contrast to other phospholipases, PLA₁ is not available on the commercial market for fine chemicals and little is known about the role of PLA₁ in cell signaling and metabolism. Received: 27 January 2000 / Received revision: 10 April 2000 / Accepted: 14 April 2000     

Polypeptide release factor eRF1 from Tetrahymena thermophila: cDNA cloning, purification and complex formation with yeast eRF3.

The first cDNA for the translational release factor eRF1 of ciliates was cloned from Tetrahymena thermophila. The coding frame contained one UAG and nine UAA codons that are reassigned for glutamine in Tetrahymena. The deduced protein sequence is 57% identical to human eRF1. The recombinant Tetrahymena eRF1 purified from a yeast expression system was able to bind to yeast eRF3 as do other yeast or mammalian eRF1s as a prerequisite step for protein termination. The recombinant Tetrahymena eRF1, nevertheless, failed to catalyze polypeptide termination in vitro with rat or Artemia ribosomes, at least in part, due to less efficient binding to the heterologous ribosomes. Stop codon specificity and phylogenetic significance of Tetrahymena eRF1 are discussed from the conservative protein feature.     

Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote

The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.     

Oral Ultrastructure and Oral Development of the Misaligned Undulating Membrane Mutant of Tetrahymena thermophila1

The misaligned undulating membrane (mum) mutant of Tetrahymena thermophila is a non-conditional, single gene recessive mutation. The major effect of the mum mutation is the production of multiple undulating membrane (UM) fragments in the oral apparatus (OA). The ultrastructure of the UM fragments of mum OAs is identical to that of the single UM of wild-type OAs. Analysis of OA development at midbody using a combination of light microscopy of protargol-stained cells and SEM of demembranated whole cells showed that the phenotypic effect of the mum mutation first becomes evident during mid to late stage 4 and is fully manifested in early stage 5. The effect of the mutation involves a proliferation of excess basal bodies in the UM field. Subsequent events in the development of the mum OA from mid to late stage 5 are identical to those in wild-type OAs. This study suggests that the mum mutation establishes conditions that allow the production of multiple UMs and thus reveals that the UM field is competent for the complete and coordinated development of several adjacent UMs. This level of regional control is not clearly evident when a single UM is present. The comparison of development of wild-type and mum OAs required an extensive reanalysis of stages 4 and 5 of normal oral development. On the basis of current and previous observations, we propose a new and more subdivided staging system for oral development in Tetrahymena.     

Enzymatic methylation of sulfide, selenide, and organic thiols by Tetrahymena thermophila

Cell extracts from the ciliate Tetrahymena thermophila catalyzed the S-adenosylmethionine-dependent methylation of sulfide. The product of the reaction, methanethiol, was detected by a radiometric assay and by a gas-chromatographic assay coupled to a sulfur-selective chemiluminescence detector. Extracts also catalyzed the methylation of selenide, and the product was shown by gas chromatography-mass spectrometry to be methaneselenol. The sulfide and selenide methyltransferase activities copurified with the aromatic thiol methyltransferase previously characterized from this organism (A.-M. Drotar and R. Fall, Pestic. Biochem. Physiol. 25:396-406, 1986), but heat inactivation experiments suggested the involvement of distinct sulfide and selenide methyltransferases. Short-term toxicity tests were carried out for sulfide, selenide, and their methylated derivatives; the monomethylated forms were somewhat more toxic than the nonmethylated or dimethylated compounds. Cell suspensions of T. thermophila exposed to sulfide, methanethiol, or their selenium analogs emitted methylated derivatives into the headspace. These results suggest that this freshwater protozoan is capable of the stepwise methylation of sulfide and selenide, leading to the release of volatile methylated sulfur or selenium gases.     

The actin gene ACT1 is required for phagocytosis, motility, and cell separation of Tetrahymena thermophila

A previously identified Tetrahymena thermophila actin gene (C. G. Cupples and R. E. Pearlman, Proc. Natl. Acad. Sci. USA 83:5160-5164, 1986), here called ACT1, was disrupted by insertion of a neo3 cassette. Cells in which all expressed copies of this gene were disrupted exhibited intermittent and extremely slow motility and severely curtailed phagocytic uptake. Transformation of these cells with inducible genetic constructs that contained a normal ACT1 gene restored motility. Use of an epitope-tagged construct permitted visualization of Act1p in the isolated axonemes of these rescued cells. In ACT1Delta mutant cells, ultrastructural abnormalities of outer doublet microtubules were present in some of the axonemes. Nonetheless, these cells were still able to assemble cilia after deciliation. The nearly paralyzed ACT1Delta cells completed cleavage furrowing normally, but the presumptive daughter cells often failed to separate from one another and later became reintegrated. Clonal analysis revealed that the cell cycle length of the ACT1Delta cells was approximately double that of wild-type controls. Clones could nonetheless be maintained for up to 15 successive fissions, suggesting that the ACT1 gene is not essential for cell viability or growth. Examination of the cell cortex with monoclonal antibodies revealed that whereas elongation of ciliary rows and formation of oral structures were normal, the ciliary rows of reintegrated daughter cells became laterally displaced and sometimes rejoined indiscriminately across the former division furrow. We conclude that Act1p is required in Tetrahymena thermophila primarily for normal ciliary motility and for phagocytosis and secondarily for the final separation of daughter cells.