DNA binding proteins from Tetrahymena thermophila

We have used DNA-cellulose chromatography to isolate single-strand binding proteins from Tetrahymena thermophila. Three major proteins which bind to denatured DNA-cellulose were obtained. The predominant protein has a molecular weight of 20 000 in sodium dodecyl sulfate - polyacrylamide gel electrophoresis and possesses many of the properties of the helix destabilizing proteins isolated from prokaryotic and eukaryotic sources. The protein facilitates denaturation of the synthetic copolymer poly[d(A-T).d(A-T)], depressing the melting temperature by nearly 40 degrees C. It also permits the renaturation of poly[d(A-T)].d(A-T)] in high salt concentration. Two other binding proteins have molecular weight of 25 000 and 23 000 in sodium dodecyl sulfate - polyacrylamide gel electrophoresis. The protein with a molecular weight of 25 000 is probably the "M protein" previously isolated from Tetrahymena thermophila which has been shown to stimulate Tetrahymena DNA polymerase. These two proteins failed to show helix destabilizing, DNA dependent ATPase, or deoxyribonuclease activities. These three proteins are abundant in the cell with approximately 1.0 x 10(6) to 10.0 x 10(6) molecules of each protein monomer per cell. One molecule of each protein monomer binds to 7 to 10 nucleotides as detected by a nitrocellulose filter binding assay. Peptide mapping of the three proteins suggests that they are all distinct. We have also found that the binding proteins can interact with Tetrahymena DNA polymerase and some other proteins to form an enzyme complex, a putative replication complex.     

Tetrahydroxystearic acid-containing taurolipid isolated from Tetrahymena thermophila

The second taurine-containing lipid (taurolipid B) was found in cells of Tetrahymena thermophila. The lipid accounted for about 1.4% of the total lipids of the cells. The lipid was subjected to mild alkaline and methanolic hydrochloric acid hydrolyses, and the structures of the hydrolysis products were identified by mass and nuclear magnetic resonance spectrometry, as taurine, 2,3,7,13-tetrahydroxystearicacid and non-hydroxy fatty acids. By spin-decoupling analysis in nuclear magnetic resonance spectrometry, the structure of the taurolipid B was identified as 2-(3-acyloxy-2,7,13-trihydroxyoctadecanoyl)aminoethane-sulfonic acid. This structure shows that taurolipid B is a homologue of the first taurine-containing lipid (taurolipid A).     

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.     

Molecular cloning and characterization of profilin from tobacco (Nicotiana tabacum): increased profilin expression during pollen maturation

Profilin has recently been identified as an actin-binding protein in higher plants. A cDNA coding for tobacco profilin, which shared an average sequence identity of 75% with other plant profilins, was isolated from a tobacco pollen cDNA library by antibody screening. Tobacco profilin was expressed in Escherichia coli and purified by affinity to poly-(L-proline) Sepharose. A rabbit antiserum was raised against recombinant tobacco profilin and used to estimate the amount of profilin expressed in different tobacco tissues. Profilin can be detected in different somatic tissues, but the expression is 50–100 fold higher in mature pollen. Immunofluorescence and confocal laser scanning microscopy showed a homogeneous distribution of profilin in the cytoplasm of in vitro cultured pollen grains and pollen tubes of tobacco whereas some growing pollen tubes were stained more intensively a their tip. A possible role of pollen profilin as a developmentally upregulated microfilament precursor in mature pollen is discussed.     

Single-cell isolation and cloning of Tetrahymena thermophila cells with a fluorescence-activated cell sorter

We developed a method for cloning cells of the ciliate Tetrahymena thermophila in chemically defined medium (CDM) using a fluorescence-activated cell sorter (FACS). Although T. thermophila is a model unicellular eukaryote, two major technical difficulties remain in its cloning. First, T. thermophila fails to proliferate from low density in CDM, particularly if the inoculum contains single cells. Second, general cloning methods are time consuming and have low throughput. Here, we modified the CDM by addition of bovine serum albumin that helped growth from an inoculum with a density of 10 cell/ml (1 cell/100 μl). In addition, we applied a FACS for isolation of single cells. We showed that it is possible to separate cell populations based on the presence or absence of phagocytosed fluorescent beads and to isolate single cells in a modified CDM by FACS. Our techniques allow the direct isolation of single cells and facilitate the establishment of clonal strains.     

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.     

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.     

Purification and characterization of Tetrahymena profilin

We subjected Tetrahymena cell extract to a poly(L-proline) affinity column for isolating profilin and obtained a protein of 12.8 kDa. Purified 12.8 kDa protein dose-dependently inhibited the polymerization of Tetrahymena actin more strongly than that of rabbit skeletal muscle actin. Because the 12.8 kDa protein fulfills properties common to profilins, the protein is considered to be Tetrahymena profilin. The present paper is the first report of the isolation of an actin-binding protein from Tetrahymena.     

The primary structure of Tetrahymena profilin

The cDNA of Tetrahymena profilin was cloned and sequenced. The deduced product has a molecular mass of 16,785 Da which is the largest among profilins known so far. Tetrahymena profilin shows higher homologies with lower eukaryotic profilins than with mammalian profilins. Although the homologies with mammalian and lower eukaryotic profilins are only 20-29% which is the lowest one among lower eukaryotic profilins, the N- and C-terminal regions of Tetrahymena profilin are considerably conserved as those in other profilins, suggesting that these regions are responsible for the essential properties common to profilins.     

New Axonemal Dynein Heavy Chains from Tetrahymena thermophila

Two dyneins can be extracted from Tetrahymena ciliary axonemes. The 22S dynein contains three heavy chains (HC), sediments at 22S in a sucrose gradient, and makes up the outer arms. The 14S dynein contains two to six HCs, sediments at 14S, and is thought to contribute to formation of the inner arms. We have identified two large proteins that are extracted from Tetrahymena axonemes with high salt and that sediment together at approximately 18S. The two large proteins cleave when subjected to UV light in the presence of ATP and vanadate, suggesting both proteins are dynein HC. Antibodies against one of the 18S HCs do not recognize 22S dynein HCs. Antibodies to 22S dynein HC do not bind appreciably to 18S dynein photocleavage fragments. Taken together, these results indicate that the large proteins that sediment at 18S are axonemal dynein heavy chains.     

Immobilization Antigens from Tetrahymena thermophila Are Glycosyl-Phosphatidylinositol-Linked Proteins

We have studied four strains of Tetrahymena thermophila , each of which expresses a different allele of the SerH gene and produces a distinctive surface protein of the immobilization antigen (i-antigen) class. Following exposure of the strains to [³H]ethanolamine or [³H]myristic acid, a protein corresponding in molecular mass to the characteristic i-antigen for that strain became highly labeled, as determined by mobility in sodium dodecylsulfate-polyacrylamide eiectrophoresis gels. Furthermore, antibodies raised to the i-antigens of the T. thermophila strains selectively immunoprecipitated radioactive proteins having molecular mass identical to that of the i-antigen characteristic for that particular strain. The lipid moieties labeled by [³H]myristate were not susceptible to hydrolysis by exogenous phosphatidylinositol-specific phospholipase C from bacteria. However, when protein extraction was carried out in the absence of phospholipase C inhibitors, radioactive fatty acids derived from [³H]myristate were rapidly cleaved from the putative i-antigens. On the basis of available data, it was concluded that T. thermophila i-antigens contain covalently-Iinked glycosyl-phospha-tidylinositol anchors.     

The Tetrahymena thermophila phagosome proteome

In vertebrates, phagocytosis occurs mainly in specialized cells of the immune system and serves as a primary defense against invading pathogens, but it also plays a role in clearing apoptotic cells and in tissue remodeling during development. In contrast, unicellular eukaryotes, such as the ciliate Tetrahymena thermophila, employ phagocytosis to ingest and degrade other microorganisms to meet their nutritional needs. To learn more about the protein components of the multistep process of phagocytosis, we carried out an analysis of the Tetrahymena phagosome proteome. Tetrahymena cells were fed polystyrene beads, which allowed for the efficient purification of phagosomes. The protein composition of purified phagosomes was then analyzed by multidimensional separation coupled with tandem mass spectrometry. A total of 453 peptides were identified that resulted in the identification of 73 putative phagosome proteins. Twenty-eight of the proteins have been implicated in phagocytosis in other organisms, indicating that key aspects of phagocytosis were conserved during evolution. Other identified proteins have not previously been associated with phagocytosis, including some of unknown function. Live-cell confocal fluorescence imaging of Tetrahymena strains expressing green fluorescent protein-tagged versions of four of the identified phagosome proteins provided evidence that at least three of the proteins (including two with unknown functions) are associated with phagosomes, indicating that the bulk of the proteins identified in the analyses are indeed phagosome associated.     

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%.     

Isolation of a Stearoyl CoA Desaturase from Tetrahymena thermophila

Cell free preparations of Tetrahymena thermophila contain an enzyme that catalyzes the direct desaturation of stearoyl CoA to octadecenoic acid. The enzyme is associated with the microsomal fraction of the ciliate. Substrate for the enzyme consists of either free stearic acid or stearoyl CoA. Both ATP and CoA are required when free stearate is the substrate and are also highly stimulatory when stearoyl CoA is the substrate. With stearoyl CoA as the substrate, either NADH or NADPH are required for desaturase activity. In the presence of ATP and CoA, either NAD or NADP can replace NADH and NADPH. Desaturase activity is optimal when the enzyme is incubated at a pH of 7.2 and a temperature of 30–35°C. Highest levels of the stearoyl CoA desaturase are found in stationary phase ciliates grown at 35°C.     

Multiple introns in a conjugation-specific gene from Tetrahymena thermophila

Multiple introns have been found in a gene from a ciliated protozoan. This Tetrahymena thermophila gene (cnjB) is large (7.5 kb mRNA) and active only during conjugation, the organism's sexual cycle. Six introns ranging in size from 62 bp to 676 bp were found when we sequenced a 3.1 kb segment of the cnjB gene together with its corresponding cDNA. We estimate, by extrapolation of our current data, a total of approximately 30 introns in this gene with a total gene size (introns plus exons) of 15 kb or more. The number of introns is surprising given the scarcity of introns in ciliate genes examined to date. Our findings constitute the first example of multiple introns in a ciliate gene. Having the sequence of several introns has allowed us to construct consensus sequences for T. thermophila mRNA introns. The 5' and 3' intron junctions resemble those of general nuclear mRNA (GT/AG rule is followed) but differences are seen. In particular, stretches of 10 or more adenines and thymines are found adjacent to the conserved GT and AGs at the junctions. Unusual aspects of the coding region of this gene are discussed.