Isolation and Identification of Specific Cortical Proteins in Tetrahymena pyriformis strain GL*†

SYNOPSIS. Pellicles of the ciliate Tetrahymena pyriformis strain GL (phenoset A) were isolated by a new procedure. Oral apparatuses were also purified by a modification of a previous method. Both preparations were characterized by electron microscopy. Proteins of the isolates were separated by analytical SDS polyacrylamide gel electrophoresis. The isolated pellicles, which included oral apparatuses, contained only 6 major proteins (gel bands), designated A through F. Bands A, B, and C, were found in the pellicle fraction, but not in the oral apparatus fraction. Therefore, these proteins are believed to be present in the somatic cortex of Tetrahymena. Bands D and E were greatly enriched in the oral apparatus fraction; these proteins are therefore believed to be present primarily in the oral apparatus. Band F, identified as tubulin, was present in both preparations. Molecular weight determinations and some selective solubilization experiments are also presented.     

Tetrahymena pyriformis as a Model System for Membrane Studies*†

SYNOPSIS. Tetrahymena pyriformis is an exceptionally useful subject for studying metabolic interrelationships among intracellular membranes. Its advantages include the striking differences in lipid composition among the cell's various functionally distinct membrane systems, indicating a pronounced lipid specificity at the membrane sites. The magnitude of these differences permits analysis of the mechanisms underlying the specificity. Even more valuable is the unique physical isolation of ciliate surface membranes from the cytoplasm of the cell. In contrast to the almost immediate equilibration of newly made lipids with preexisting lipids found in most cells, Tetrahymena surface membranes have a lipid turnover slow enough to be conveniently analyzed. Finally, the well-studied responses of Tetrahymena to such physiological stresses as heat and starvation may be used to evaluate the effects of environmental factors on membrane formation.     

Transformation in Tetrahymena pyriformis: Description of an Inducible Phenotype*†

SYNOPSIS. Transformation of Tetrahymena pyriformis to a rapid-swimming (presumably dispersal) form can be induced by washing cells and suspending them in distilled H₂O, Dryl's solution or 10 mm Tris. Transformation is possible with high efficiency in mass cultures of axenically grown cells within ～ 5 h at 30 C. The radically different phenotype produced during transformation is characterized by a more elongate body form, increased numbers of somatic basal bodies and cilia, a long caudal cilium and oral membranelles positioned beneath the cell surface. DNA quantities characteristic of G1, S, and G2 cells are found in these transformed ciliates, suggesting that achievement of a particular stage in the DNA-division cycle is not a prerequisite for transformation. Preliminary observations on cells belonging to syngens 2–12 indicate that they also have a capacity to form a caudal cilium, but that the amicronucleate strain GL-C does not. Possible relevance of the transformed phenotype for taxonomy of Tetrahymena is discussed.     

Studies of Membrane Formation in Tetrahymena pyriformis. VIII. On the Origin of Membranes Surrounding Food Vacuoles*†

SYNOPSIS. A procedure was devised for the isolation of purified food vacuoles from Tetrahymena pyriformis fed particles of ferric oxide. Phospholipids extracted from vacuolar membranes were more similar in composition to the lipids of microsomes than to lipids of whole cells, cilia or post-microsomal supernatant. Fractionation of cells grown in the presence of [¹⁴C]palmitic acid or [³²P]inorganic phosphate also revealed similarities in the specific radioactivities of microsomes and vacuolar membranes. The data suggested that vacuolar membranes arise from a pool of cytoplasmic membranes.     

Poly(A)+ RNA from Tetrahymena: Stimulation of Protein Synthesis in Vitro*†

SYNOPSIS Cell-free synthesis of high molecular weight polypeptides, programmed by RNA from Tetrahymena pyriformis strain W is reported, and methods for preparation of the RNA are described. The RNA was extracted by the SDS-phenol-chloroform-isoamyl alcohol technic. The bulk of extracted RNA was ribosomal and on sucrose gradients peaked at -17S and 25S. After heat denaturation all the 25S RNA was converted to 17S. indicating the presence of hidden breaks, possibly the result of nuclease activity during extraction. Nevertheless, when poly(A)–RNA was collected using oligo-(dT)-cellulose column chromatography, it promoted a 15–fold increase in incorporation of [35S] methionine into TCA-precipitable material. Slab-gel electrophoresis and autoradiography of the product revealed 12 different major polypeptides, varying in weight from 28.000 to 65,000 Daltons. A method for preparation of translatable RNA from Tetrahymena will make possible the comparison of messenger RNAs associated with specific cell structures and with different developmental events.     

Studies on the Conditions Required for Optimum Recovery of Tetrahymena pyriformis Strain S (Phenoset A) after Freezing to and Thawing from –196 C

The toxicity of several cryoprotective agents was tested at room temperature (23 C) against Tetrahymena pyriformis strain S (Phenoset A) at different stages of the growth cycle. Polyvinylpyrrolidone (PVP-40) at 10% (v/v) concentration was without effect at any stage in the growth cycle, while 1.2 M glycerol immobilized the cells which were disrupted very shortly afterwards. The toxicity of 0.25 M glucose was largely independent of the position of the cells in the growth cycle, but the toxicity of 0.25 M sucrose and 1.4 M dimethylsulfoxide (DMSO) was most marked in late log- and stationary-phase cells. After log-phase cells had been equilibrated with 1.4 M DMSO for 1 hr, the number of cells surviving cooling at defined rates from 0.45 to 12 C/min decreased as the final temperature decreased from –30 to –60 C. A temperature of –53 C was found to be the optimum from which cells cooled at a given rate could be cooled rapidly to –196 C. Nevertheless, when cells were cooled at defined rates to –35, –45, or –53 C and then rapidly to –196 C the optimum rate of cooling to these temperatures was found to be 1 C/min. The optimum rate of cooling to –60 C prior to plunging into liquid nitrogen was found to be 2.7 C/min.     

A metagenomic approach for the identification and cloning of an endoglucanase from rice straw compost

Over the years, culturable cellulase-producing microorganisms have been isolated from a variety of sources and genes of cellulolytic enzymes have been cloned. Then again, the “great plate count anomaly” phenomenon necessitates a culture-independent metagenomic approach for the isolation of cellulolytic genes from microorganisms in their natural environment. We have constructed a metagenomic library derived from rice straw composts. Of 2739 clones screened, a lambda clone carrying a 12 kb genomic fragment was able to yield a clear zone on an agar plate containing carboxymethyl cellulose (CMC). A 4.7 kb subclone, generated by restriction enzyme digestion, was found to harbor a GH12 cellulase gene, RSC-EG1, encoding 464 amino acids along with two other ORFs. The identified cellulolytic gene showed more than 70% similarity on the amino acid level with cellulase from Micromonospora aurantiaca and Thermobispora sp. Interestingly, RSC-EG1 contains a stretch of approximately 86 amino acids not present in either of these close relatives. Our results demonstrated that RSC-EG1, stable over a wide temperature and pH range, is a novel endoglucanase, and provided the first example of metagenomics approach to isolate cellulolytic gene from rice straw composts.