Mutual detoxification of mercury and selenium in unicellular Tetrahymena.

Selenium (Se) is commonly recognized as a protective element with an antagonistic effect against mercury (Hg) toxicity. However, the mechanisms of this Hg-Se antagonism are complex and remain controversial. To gain insight into the Hg-Se antagonism, a type of unicellular eukaryotic protozoa (Tetrahymena malaccensis, T. malaccensis) was selected and individually or jointly exposed to two Hg and three Se species. We found that Se species showed different toxic effects on the proliferation of T. malaccensis with the toxicity following the order: selenite (Se(IV))>selenomethionine (SeMeth)>selenate (Se(VI)). The Hg-Se antagonism in Tetrahymena was observed because the joint toxicity significantly decreased under co-exposure to highly toxic dosages of Hg and Se versus individual toxicity. Unlike Se(IV) and Se(VI), non-toxic dosage of SeMeth significantly decreased the Hg toxicity, revealing the influence of the Se species and dosages on the Hg-Se antagonism. Unexpectedly, inorganic divalent Hg (Hg2+) and monomethylmercury (MeHg) also displayed detoxification towards extremely highly toxic dosages of Se, although their detoxifying efficiency was discrepant. These results suggested mutual Hg-Se detoxification in T. malaccensis, which was highly dependent on the dosages and species of both elements. As compared to other species, SeMeth and MeHg promoted the Hg-Se joint effects to a higher degree. Additionally, the Hg contents decreased for all the Hg-Se co-exposed groups, revealing a sequestering effect of Se towards Hg in T. malaccensis.

Optimization and validation of a colorimetric assay for Tetrahymena sp. survival.

A colorimetric assay based on the reduction of 3-(4,5-dimethyl-2-thiazol-2-yl)-2,5-diphenyl-2h-tetrazolium bromide (MTT), for the quantification of Tetrahymena sp. survival is described. An increase in the concentration of Tetrahymena sp. cells from 0 to 1 x 10(6) cells/ml produced a linear (R(2)=0.9965) increase in the optical density (OD, 570-630 nm), and dead cells (pre-exposed to 250 mg/l formalin for 4 h) did not produce a background reading. Cells exposed to sublethal concentrations to formalin (100 mg/l or less for 4 h) recovered their growth. Using the MTT assay, we determined that Tetrahymena sp. is sensitive to formalin, chloramine-T, hydrogen peroxide, copper sulfate and NaCl. The sensitivity increased with increasing chemical concentrations and exposure time. Tetrahymena sp. was resistant to bromex and malachite green. The use of this assay in drug screening for the development of treatments for tetrahymenosis and as a bioassay to evaluate the toxicity of environmental toxicants is discussed.

Adenylyl cyclases from Plasmodium, Paramecium and Tetrahymena are novel ion channel/enzyme fusion proteins.

In Paramecium, cAMP formation is stimulated by a potassium conductance, which is an intrinsic property of the adenylyl cyclase. We cloned a full-length cDNA and several gDNA fragments from Paramecium and Tetrahymena coding for adenylyl cyclases with a novel domain composition. A putative N-terminal ion channel domain contains a canonical S4 voltage-sensor and a canonical potassium pore-loop located C-terminally after the last transmembrane span on the cytoplasmic side. The adenylyl cyclase catalyst is C-terminally located. DNA microinjection of a green fluorescent protein (GFP)-tagged construct into the macronucleus of Paramecium resulted in ciliary localization of the expressed protein. An identical gene coding for an ion-channel adenylyl cyclase was cloned from the malaria parasite Plasmodium falciparum. Expression of the catalytic domain of the latter in Sf9 cells yielded an active homodimeric adenylyl cyclase. The occurrence of this highly unique subtype of adenylyl cyclase appears to be restricted to ciliates and apicomplexa.

Tetrahymena fimbrin localized in the division furrow bundles actin filaments in a calcium-independent manner.

In cytokinesis, the contractile ring constricts the cleavage furrow. However, the formation and properties of the contractile ring are poorly understood. Fimbrin has two actin-binding domains and two EF-hand Ca(2+)-binding motifs. Ca(2+) binding to the EF-hand motifs inhibits actin-binding activity. In Tetrahymena, fimbrin is localized in the cleavage furrow during cytokinesis. In a previous study, Tetrahymena fimbrin was purified with an F-actin affinity column. However, the purified Tetrahymena fimbrin was broken in to a 60 kDa fragment of a 70 kDa full length fimbrin. In this study, we investigated the properties of recombinant Tetrahymena fimbrin. In an F-actin cosedimentation assay, Tetrahymena fimbrin bound to F-actin and bundled it in a Ca(2+)-independent manner, with a K(d) of 0.3 micro M and a stoichiometry at saturation of 1:1.4 (Tetrahymena fimbrin: actin). In the presence of 1 molecule of Tetrahymena fimbrin to 7 molecules of actin, F-actin was bundled. Immunofluorecence microscopy showed that a dotted line of Tetrahymena fimbrin along the cleavage furrow formed a ring structure. The properties and localization of Tetrahymena fimbrin suggest that it bundles actin filaments in the cleavage furrow and plays an important role in contractile ring formation during cytokinesis.

Stability of intramolecular DNA quadruplexes: comparison with DNA duplexes.

We have determined the stability of intramolecular quadruplexes that are formed by a variety of G-rich sequences, using oligonucleotides containing appropriately placed fluorophores and quenchers. The stability of these quadruplexes is compared with that of the DNA duplexes that are formed on addition of complementary C-rich oligonucleotides. We find that the linkers joining the G-tracts are not essential for folding and can be replaced with nonnucleosidic moieties, though their sequence composition profoundly affects quadruplex stability. Although the human telomere repeat sequence d[G(3)(TTAG(3))(3)] folds into a quadruplex structure, this forms a duplex in the presence of the complementary C-rich strand at physiological conditions. The Tetrahymena sequence d[G(4)(T(2)G(4))(3)], the sequence d[G(3)(T(2)G(3))(3)], and sequences related to regions of the c-myc promoter d(G(4)AG(4)T)(2) and d(G(4)AG(3)T)(2) preferentially adopt the quadruplex form in potassium-containing buffers, even in the presence of a 50-fold excess of their complementary C-rich strands, though the duplex predominates in the presence of sodium. The HIV integrase inhibitor d[G(3)(TG(3))(3)] forms an extremely stable quadruplex which is not affected by addition of a 50-fold excess of the complementary C-rich strand in both potassium- and sodium-containing buffers. Replacing the TTA loops of the human telomeric repeat with AAA causes a large decrease in quadruplex stability, though a sequence with AAA in the first loop and TTT in the second and third loops is slightly more stable.

Quantitative structure-activity relationships for the inhibition toxicity to root elongation of Cucumis sativus of selected phenols and interspecies correlation with Tetrahymena pyriformis.

The comparative toxicities of selected phenols to higher plants Cucumis sativus were measured and the negative logarithm molar concentration of the root elongation median inhibition (IRC50) were derived. Quantitative structure-activity relationships (QSARs) were developed to explore the toxicity influencing factors and for predictive purpose. The toxicity data, fell into two classes: polar narcosis and bio-reactive. For polar narcotic phenols, a highly significant two-parameter QSAR based on 1-octanol/water partition coefficient (logKow) and energy of the lowest unoccupied orbital (E(lumo)) was derived (IRC50 = 0.77 log Kow - 0.39E(lumo) + 2.36 n = 22 r2 = 0.89). The five bio-reactive chemicals proved to show elevated toxicity due to their typical substructure involved diverse reactive mechanisms. In an effort to model all chemicals, a robust multiple-variable QSAR combining logKow, E(lumo) and Qmax, the most negative net atomic charge, was developed (IRC50 = 0.65 logKow - 0.72E(lumo) + 0.23Qmax + 2.81 n = 27 r2 = 0.94), indicating that hydrophobicity, electrophilicity and hydrogen bond interaction contribute mainly to the phytotoxicity. The toxicological data was compared with Tetrahymena pyriformis 2-d population growth inhibition toxicity (IGC50) and excellent interspecies correlations were observed both for the polar narcotics and for five reactive chemicals (for polar narcotics: IRC50 = 0.95IGC50 + 1.07 n = 16 r2 = 0.89; for bio-reactive chemicals: IRC50 = 0.98IGC50 + 2.19 n = 5 r2 = 0.97; and for all: IRC50 = 0.93IGC50 + 1.63 n = 21 r2 = 0.87). This suggested that T pyriformis toxicity could serve as a surrogate of C. sativus toxicity for phenols and interspecies correlation also could be established for reactive chemicals.

Identification, cloning and characterisation of a novel copper-metallothionein in tetrahymena pigmentosa. Sequencing of cDNA and expression.

The protist Tetrahymena pigmentosa accumulates large amounts of metal ions, particularly cadmium and copper. This capability is linked to the induction of metallothioneins (MTs), cysteine-rich metal-binding proteins found in protists, plants and animals. The present study focuses on a novel inducible MT-isoform isolated from Tetrahymena after exposure to a non-toxic dose of copper. The cDNA sequence was determined utilising the partial peptide sequence of purified protein. The Cu-MT cDNA encodes 96 amino acids containing 28 cysteine residues (29%) arranged in motifs characteristic of the metal-binding regions of vertebrate and invertebrate MTs. Both the amino acid and nucleotide sequences differ, not only from other animal MTs, but also from the previously characterised Tetrahymena Cd-MT. Both MTs contain the structural pattern GTXXXCKCXXCKC, which may be proposed as a conservative sequence of Tetrahymena MTs. Cu-dependent regulation of MT expression was also investigated by measuring MT-mRNA and MT levels. MT synthesis occurs very quickly and MT contents increase with Cu accumulation. The induction of Cu-MT mRNA is very rapid, with no observable lag period, and is characterised by transient fluctuation, similar to that described for Cd-MT mRNA. The data reported here indicate that, also in the unicellular organism Tetrahymena, two very different MT isoforms, which perform different biological functions, are expressed according to the inducing metal, Cu or Cd.

Cloning and biochemical analysis of the tetrahymena origin binding protein TIF1: competitive DNA binding in vitro and in vivo to critical rDNA replication determinants.

Cis-acting type I elements regulate the initiation of DNA replication, replication fork movement, and transcription of the Tetrahymena thermophila rDNA minichromosome and are required for cell cycle-controlled replication and developmentally programmed gene amplification. Previous studies identified three in vitro single-stranded type I element binding activities that were proposed to play distinct roles in replication control. Here we describe the cloning of one of these genes, TIF1, and we provide evidence for its association with type I elements in vivo. Furthermore, we show that TIF1 interacts (in vitro and in vivo) with pause site elements (PSE), which co-localize with replication initiation and fork arrest sites, and are shown to be essential. The in vivo accessibility of PSE and type I elements to potassium permanganate suggests that origin regions are frequently unwound in native chromatin. TIF1 contains sequence similarity to the Solanum tuberosum single strand-specific transcription factor, p24, and a related Arabidopsis protein. Antisense inhibition studies suggest that TIF1 competes with other proteins for PSE and type I element binding. TIF1 displays a marked strand bias in vivo, discriminating between origin- and promoter-proximal type I elements. We propose that this bias selectively modulates the binding of a different subset of proteins to the respective regulatory elements.

An unconventional origin of metal-ion rescue and inhibition in the Tetrahymena group I ribozyme reaction.

The presence of catalytic metal ions in RNA active sites has often been inferred from metal-ion rescue of modified substrates and sometimes from inhibitory effects of alternative metal ions. Herein we report that, in the Tetrahymena group I ribozyme reaction, the deleterious effect of a thio substitution at the pro-Sp position of the reactive phosphoryl group is rescued by Mn2+. However, analysis of the reaction of this thio substrate and of substrates with other modifications strongly suggest that this rescue does not stem from a direct Mn2+ interaction with the Sp sulfur. Instead, the apparent rescue arises from a Mn2+ ion interacting with the residue immediately 3' of the cleavage site, A(+1), that stabilizes the tertiary interactions between the oligonucleotide substrate (S) and the active site. This metal site is referred to as site D herein. We also present evidence that a previously observed Ca2+ ion that inhibits the chemical step binds to metal site D. These and other observations suggest that, whereas the interactions of Mn2+ at site D are favorable for the chemical reaction, the Ca2+ at site D exerts its inhibitory effect by disrupting the alignment of the substrates within the active site. These results emphasize the vigilance necessary in the design and interpretation of metal-ion rescue and inhibition experiments. Conversely, in-depth mechanistic analysis of the effects of site-specific substrate modifications can allow the effects of specific metal ion-RNA interactions to be revealed and the properties of individual metal-ion sites to be probed, even within the sea of metal ions bound to RNA.

What do linker histones do in chromatin?

Knockout experiments in Tetrahymena show that linker histone H1 is not essential for nuclear assembly or cell viability. These results, together with a series of biochemical and cell biological observations, challenge the existing paradigm that requires linker histones to be a key organizing component of higher-order chromatin structure. The H1 knockouts also reveal a much more subtle role for H1. Instead of acting as a general transcriptional repressor, H1 is found to regulate a limited number of specific genes. Surprisingly, H1 can both activate and repress transcription. We discuss how this architectural protein might accomplish this important regulatory role.

Solution structure of the Tetrahymena telomeric repeat d(T2G4)4 G-tetraplex.

BACKGROUND: Telomeres in eukaryotic organisms are protein-DNA complexes which are essential for the protection and replication of chromosomal termini. The telomeric DNA of Tetrahymena consists of T2G4 repeats, and models have been previously proposed for the intramolecular folded structure of the d(T2G4)4 sequence based on chemical footprinting and cross-linking data. A high-resolution solution structure of this sequence would allow comparison with the structures of related G-tetraplexes. RESULTS: The solution structure of the Na(+)-stabilized d(T2G4)4 sequence has been determined using a combined NMR-molecular dynamics approach. The sequence folds intramolecularly into a right-handed G-tetraplex containing three stacked G-tetrads connected by linker segments consisting of a G-T-T-G lateral loop, a central T-T-G lateral loop and a T-T segment that spans the groove through a double chain reversal. The latter T-T connectivity aligns adjacent G-G-G segments in parallel and introduces a new G-tetraplex folding topology with unprecedented combinations of strand directionalities and groove widths, as well as guanine syn/anti distributions along individual strands and around individual G-tetrads. CONCLUSIONS: The four repeat Tetrahymena and human G-tetraplexes, which differ by a single guanine for adenine substitution, exhibit strikingly different folding topologies. The observed structural polymorphism establishes that G-tetraplexes can adopt topologies which project distinctly different groove dimensions, G-tetrad base edges and linker segments for recognition by, and interactions with, other nucleic acids and proteins.

Kinetic intermediates in RNA folding.

The folding pathways of large, highly structured RNA molecules are largely unexplored. Insight into both the kinetics of folding and the presence of intermediates was provided in a study of the Mg(2+)-induced folding of the Tetrahymena ribozyme by hybridization of complementary oligodeoxynucleotide probes. This RNA folds via a complex mechanism involving both Mg(2+)-dependent and Mg(2+)-independent steps. A hierarchical model for the folding pathway is proposed in which formation of one helical domain (P4-P6) precedes that of a second helical domain (P3-P7). The overall rate-limiting step is formation of P3-P7, and takes place with an observed rate constant of 0.72 +/- 0.14 minute-1. The folding mechanism of large RNAs appears similar to that of many multidomain proteins in that formation of independently stable substructures precedes their association into the final conformation.

Three Tetrahymena tRNA(Gln) isoacceptors as tools for studying unorthodox codon recognition and codon context effects during protein synthesis in vitro.

Three glutamine tRNA isoacceptors are known in Tetrahymena thermophila. One of these has the anticodon UmUG which reads the two normal glutamine codons CAA and CAG, whereas the two others with CUA and UmUA anticodons recognize UAG and UAA, respectively, which serve as termination codons in other organisms. We have employed these tRNA(Gln)-isoacceptors as tools for studying unconventional base interactions in a mRNA- and tRNA-dependent wheat germ extract. We demonstrate here (i) that tRNA(Gln)UmUG suppresses the UAA as well as the UAG stop codon, involving a single G:U wobble pair at the third anticodon position and two simultaneous wobble base pairings at the first and third position, respectively, and (ii) that tRNA(Gln)CUA, in addition to its cognate codon UAG, reads the UAA stop codon which necessitates a C:A mispairing in the first anticodon position. These unorthodox base interactions take place in a codon context which favours readthrough in tobacco mosaic virus (TMV) or tobacco rattle virus (TRV) RNA, but are not observed in a context that terminates zein and globin protein synthesis. Furthermore, our data reveal that wobble or mispairing in the middle position of anticodon-codon interactions is precluded in either context. The suppressor activities of tRNAs(Gln) are compared with those of other known naturally occurring suppressor tRNAs, i.e., tRNA(Tyr)G psi A and tRNA(Trp)CmCA. Our results indicate that a 'leaky' context is neither restricted to a single stop codon nor to a distinct tRNA species.

Monovalent cation induced structural transitions in telomeric DNAs: G-DNA folding intermediates.

Telomeric DNA consists of G- and C-rich strands that are always polarized such that the G-rich strand extends past the 3' end of the duplex to form a 12-16-base overhang. These overhanging strands can self-associate in vitro to form intramolecular structures that have several unusual physical properties and at least one common feature, the presence of non-Watson-Crick G.G base pairs. The term "G-DNA" was coined for this class of structures (Cech, 1988). On the basis of gel electrophoresis, imino proton NMR, and circular dichroism (CD) results, we find that changing the counterions from sodium to potassium (in 20 mM phosphate buffers) specifically induces conformational transitions in the G-rich telomeric DNA from Tetrahymena, d(T2G4)4 (TET4), which results in a change from the intramolecular species to an apparent multistranded structure, accompanied by an increase in the melting temperature of the base pairs of greater than 25 degrees, as monitored by loss of the imino proton NMR signals. NMR semiselective spin-lattice relaxation rate measurements and HPLC size-exclusion chromatography studies show that in 20 mM potassium phosphate (pH 7) buffer (KP) TET4 is approximately twice the length of the form obtained in 20 mM sodium phosphate (pH 7) buffer (NaP) and that mixtures of Na+ and K+ produce mixtures of the two forms whose populations depend on the ratio of the cations. Since K+ and NH4+ are known to stabilize a parallel-stranded quadruplex structure of poly[r(I)4], we infer that the multistranded structure is a quadruplex. Our results indicate that specific differences in ionic interactions can result in a switch in telomeric DNAs between intramolecular hairpin-like or quadruplex-containing species and intermolecular quadruplex structures, all of which involve G.G base pairing interactions. We propose a model in which duplex or hairpin forms of G-DNA are folding intermediates in the formation of either 1-, 2-, or 4-stranded quadruplex structures. In this model monovalent cations stabilize the duplex and quadruplex forms via two distinct mechanisms, counterion condensation and octahedral coordination to the carbonyl groups in stacked planar guanine "quartet" base assemblies. Substituting one of the guanosine residues in each of the repeats of the Tetrahymena sequence to give the human telomeric DNA, d(T2AG3)4, results in less effective K(+)-dependent stabilization. Thus, the ion-dependent stabilization is attenuated by altering the sequence. Upon addition of the Watson-Crick (WC) complementary strand, only the Na(+)-stabilized structure dissociates quickly to form a WC double helix.(ABSTRACT TRUNCATED AT 400 WORDS)

Visualizing the higher order folding of a catalytic RNA molecule.

The higher order folding process of the catalytic RNA derived from the self-splicing intron of Tetrahymena thermophila was monitored with the use of Fe(II)-EDTA-induced free radical chemistry. The overall tertiary structure of the RNA molecule forms cooperatively with the uptake of at least three magnesium ions. Local folding transitions display different metal ion dependencies, suggesting that the RNA tertiary structure assembles through a specific folding intermediate before the catalytic core is formed. Enzymatic activity, assayed with an RNA substrate that is complementary to the catalytic RNA active site, coincides with the cooperative structural transition. The higher order RNA foldings produced by Mg(II), Ca(II), and Sr(II) are similar; however, only the Mg(II)-stabilized RNA is catalytically active. Thus, these results directly demonstrate that divalent metal ions participate in general folding of the ribozyme tertiary structure, and further indicate a more specific involvement of Mg(II) in catalysis.

The effect of dietary sterol on the activity of fatty acid desaturases isolated from Tetrahymena setosa.

Tetrahymena setosa has a nutritional requirement for micro amounts of sterol, a requirement which is also satisfied by relatively large amounts of either intact phospholipids or a mixture of unsaturated fatty acids normally found in these ciliates. Three microsomal fatty acyl-CoA desaturases have been isolated from T. setosa and partially characterized. These enzymes which can account for the formation of the majority of the ciliate's unsaturated fatty acids, include: a delta 9, a delta 12 and a delta 6 desaturase which catalyze the transformation of stearoyl-CoA to oleic acid, of oleoyl-CoA to linoleic acid and of linoleoyl-CoA to gamma-linolenic acid, respectively. The stearoyl CoA desaturase required NAD (or NADP), ATP and free CoA; the delta 6 and delta 12 desaturases required NADP, but not ATP or CoA. Cellular levels of the three desaturases were highest in mid-logarithmic phase cells and lowest in stationary phase cells. In order to determine if there was a relationship between the sterol requirement and the ability of the organism to desaturate, T. setosa was grown in a synthetic medium supplemented with either cholesterol or a phospholipid which permits growth in the absence of cholesterol, or with both phospholipid and cholesterol. Cells grown with phospholipid alone had only half as much stearoyl-CoA and oleoyl-CoA desaturase activity as cells of identical culture age grown either on cholesterol alone or on cholesterol plus phospholipid.

Characterization of the mode of binding of substrates to the active site of Tetrahymena self-splicing RNA using 5-fluorouracil-substituted mini-exons.

Splice-site selection specificity in Tetrahymena self-splicing RNA is thought to be mediated by a base-paired complex between a CUCUCU sequence on the end of the 5' exon and a GGGAGG guide sequence in the intron. The substitution of uracil (U) in oligonucleotide mini-exons with 5-fluorouracil (UF), an analogue bearing a much more acidic N-3 proton, allowed us to test the role of hydrogen bonding between complementary bases in the splice-site selection process. The affinities of (U) and (UF) mini-exons for the ribozyme active site were similar and several orders of magnitude greater than expected from base pairing alone. In contrast to CUCU, the CUFCUF mini-exon lost substrate activity with increasing pH, presumably due to ionization of the UF residues. However, the apparent pK values of these residues were several pK units above that of free UF, indicating that the mini-exon is shielded from the solvent by an active site of low polarity. Loss of the pyrimidine N-3 hydrogen bond by selective ionization of the UF residues decreased the binding of CUFCUF to the ribozyme only 3-fold but did prevent its ligation to the 3' exon. Temperature dependence of substrate activity was identical for both (U) and (UF) mini-exons, whereas the UF-substituted ribozyme lost activity at a considerably lower temperature than did the natural (U) ribozyme. These observations indicate that hydrogen-bonded base pairs involving the U residues contribute little to the total binding energy of the 5' splice site with the active site of the ribozyme, but probably help to align the splice sites properly for ligation.

Stereochemistry of RNA cleavage by the Tetrahymena ribozyme and evidence that the chemical step is not rate-limiting.

The intervening sequence of the ribosomal RNA precursor of Tetrahymena is a catalytic RNA molecule, or ribozyme. Acting as a sequence-specific endoribonuclease, it cleaves single-stranded RNA substrates with concomitant addition of guanosine. The chemistry of the reaction has now been studied by introduction of a single phosphorothioate in the substrate RNA at the cleavage site. Kinetic studies show no significant effect of this substitution on kcat (rate constant) or Km (Michaelis constant), providing evidence that some step other than the chemical step is rate-limiting. Product analysis reveals that the reaction proceeds with inversion of configuration at phosphorus, consistent with an in-line, SN2 (P) mechanism. Thus, the ribozyme reaction is in the same mechanistic category as the individual displacement reactions catalyzed by protein nucleotidyltransferases, phosphotransferases, and nucleases.

Stereochemical course of catalysis by the Tetrahymena ribozyme.

The group I intron from Tetrahymena catalyzes phosphodiester transfer reactions on various RNA substrates. A modified RNA substrate with a phosphorothioate group in one stereoisomeric form at the site of reaction was synthesized in order to determine the stereochemical course of an RNA-catalyzed reaction. The reaction product was digested with a stereospecific nuclease to determine the configuration of the product phosphorothioate. The reaction occurs with inversion of configuration at phosphorus, implying an in-line pathway for the reaction.