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BTAN (Sumi Y et al. (1982) Histochemistry 73:481) was investigated as a histochemical Cd/Zn chelator. Cd-BTAN exhibits a main peak about 635 nm, while Zn-BTAN exhibits a main peak about 644 nm. The isobestic wavelength for Cd-BTAN and Zn-BTAN is 638 nm. The microscopical detection limit for Cd is about 25 amol/microns 2, and for Zn about 5 amol/microns 2. The relation between metal and bound chelator is fairly linear at a BTAN concentration more than 10-fold the metal concentration. Histochemical localization was fair to good, with a crystal size of up to 0.2-0.3 micron. The chelate was unaffected by hydrophilic and largely also by hydrophobic mounting media. The original staining procedure proved erratic and was modified. Posttreatment with oxine to selectively demonstrate Cd in the presence of Zn (Sumi Y et al. 1982) seriously reduced the staining intensity. Post-treatment for 8-15 min with HCl, 0.5 mol/l, in 50% ethanol removed Cd-BTAN completely with little reduction of Zn staining intensity, even from sites with 5x as much Cd as Zn. It is concluded that BTAN permits direct quantitative detection of (Zn + Cd). Provided certain precautions are taken quantitative detection of Zn and quantitation of Cd in mixed Zn/Cd sites is possible by microphotometry of the stained section before and after differentiation for 8-15 min with the HCl/50% ethanol medium.

We have previously shown that actin from Tetrahymena pyriformis has a very divergent primary structure (Hirono, M., Endoh, H., Okada, N., Numata, O., & Watanabe, Y. (1987) J. Mol. Biol. 194, 181-192) and that though it shares essential properties with skeletal muscle actin, it does not interact at all with phalloidin or DNase I (Hirono, M., Kumagai, Y., Numata, O., & Watanabe, Y. (1989) Proc. Natl. Acad. Sci. U.S. 86, 75-79). In this study, we investigated the copolymerization of this actin with skeletal muscle actin by direct observation of the heteropolymers formed from the two actins by means of electron microscopy. We also examined the binding of actin-binding proteins from skeletal muscle or smooth muscle to Tetrahymena actin by means of a cosedimentation assay. The results show that (i) Tetrahymena actin copolymerizes with skeletal muscle actin and that (ii) muscle myosin subfragment 1 binds to it in the absence of ATP, like skeletal muscle actin. However, it was also shown that (iii) muscle alpha-actinin hardly binds to Tetrahymena actin and that (iv) muscle tropomyosin does not bind to it at all. The results show that Tetrahymena actin has both properties similar and dissimilar to those of skeletal muscle actin.

Histones bound to gene-sized pieces of DNA in the macronucleus of Oxytricha sp. consist of five main fractions: f1, f2a1, f2a2, f2b and f3. Although the slightly lysine-rich and the arginine-rich histones in Oxytricha macronuclei are similar to comparable fractions in vertebrates, the lysine-rich f1 fraction differs. Oxytricha f1 is unique among eukaryotes in that it migrates faster than f2a1 and is located nearest the cathode in polyacrylamide-urea gels.

We have examined the fatty acid composition of the mitochondrial membranes in three strains of Tetrahymena pyriformis. All three had similar components and exhibited large amounts of unsaturated fatty acids. The cytoplasmic mutant, CA-10, which has a slower growth rate and unusual membrane morphology, had a slightly higher amount of iso-acids but was otherwise similar to the other strains in fatty acid composition. Arachidonic acid, previously undetected in extracts of Tetrahymena, was identified as a minor component of the mitochondrial membrane.

Fluorescence cytophotometry was used to study nuclear DNA content and synthesis patterns during meiosis, fertilization and macronuclear development in the ciliated protozoon, Tetrahymena pyriformis, syngen 1. It was found that cells entered conjugation with a G1 (45C) macronucleus and a G2 (4C) micronucleus. During meiosis the micronucleus was reduced to 4 haploid nuclei, each with a 1C amount of DNA; each meiotic product then replicated to 2C, but only the nucleus next to the attachment membrane in each conjugant divided to form the two 1C gametic nuclei. The gametic nuclei replicated to 2C prior to fertilization; hence there was no S-period in the 4C fertilization nucleus (synkaryon). The first postzygotic division products immediately entered an S-period to become 4C, and at the second postzygotic division, each of the two 4C nuclei in each conjugant divided to form one 2C micronucleus and one 2C macronuclear Anlage. The macronuclear Anlagen began DNA synthesis immediately and were about 8C at the completion of conjugation; the micronuclei did not undergo rapid DNA doubling and measured between 2C and 3C when the conjugants separated. The old macronucleus did not participate in any S-period during conjugation and began to decompose after the second postzygotic division; it contained an average of 24C at the end of conjugation. From this sequence of nuclear divisions a pattern emerges that, unless a general cytoplasmic signal for DNA synthesis is suppressed, DNA synthesis always occurs in micronuclear division products immediately following separation of sister chromatids. Nuclear development continued in the first two cell cycles after conjugation. In exconjugants (the first cycle), macronuclear Anlagen underwent two rounds of DNA synthesis to become 32C and both micronuclei also underwent DNA synthesis. However, prior to the first cell division, one micronucleus and the old macronucleus completely disintegrated, and at the first cell division the remaining 4C micronucleus divided and one macronuclear Anlage was distributed to each resulting caryonide. At the end of the second cell cycle, the dividing macronucleus of each caryonide contained about 128C. These results relate to the question of ploidy of macronuclear subunits. It is argued that the G1 macronucleus contains 22 or 23 diploid subunits, each subunit being a copy of the diploid micronuclear genome. It is suggested that unequal macronuclear division relates to the question of subunit ploidy by playing a role in the phenomenon of macronuclear assortment.

Acid extracts of Tetrahymena pyriformis, a ciliated protozoan grown in defined medium revealed the presence of materials with salmon-type calcitonin immunoactivity. These findings add support to earlier reports of the presence of materials closely resembling vertebrate peptide hormones in unicellular microbes.

Antibodies directed against whole histone and purified lysine-rich histone H1 extracted from isolated macronuclei of the ciliate Tetrahymena were obtained and conjugated to fluorescein isothiocyanate. The fluorescein-antibody conjugates were used to directly label Tetrahymena cells. Both macro- and micronuclei were visibly fluorescent in cells stained with anti-whole histone conjugate. However, the anti-H1 conjugate only labeled macronuclei. This in situ demonstration of the lack of positive immunofluorescent staining of micronuclei with anti-H1 conjugate provide further evidence for the absence of H1 in the genetically inactive, mitotically dividing Tetrahymena micronucleus.

Mobility patterns of 5 isoenzymes in strains of Tetrahymena pyriformis were demonstrated using polyacrylamide disc gel electrophoresis. Six stock strains were compared in these patterns to 4 strains representative of each of the previously described 4 major "phenotypic sets." Stock strains segregated into predicted "phenosets." and essentially confirmed validity and reproducibility of such a discrimination method. The proposal that new strain designations be assigned on a basis of "phenoset" conformity is reaffirmed.

Tetrahymena outer doublet tubulin was compared with neurotubulin and Chlamydomonas flagellar tubulin on SDS-polyacrylamide gels. Tetrahymena alpha tubulin did not comigrate with either brain or flagellar alpha tubulins, although brain, flagellar, and ciliary beta tubulins all comigrated. Axonemal tubulin from Tetrahymena strain ST was compared with this tubulin from strains W, S, HSM, and E, and all were found to have the same mobilities. Poly-A containing RNA was separated from whole cell Tetrahymena RNA by oligo-dT cellulose chromatography. Poly-A+ RNA from 24-h cultures (early exponential growth) stimulated greater incorporation of amino acids into polypeptides in the wheat germ cell-free translation system than did poly-A+ RNA from 36-h and 49-h cultures. When separated on SDS-polyacrylamide gels, the translation products of the 24-h poly-A+ RNA had 2 prominent protein bands which comigrated with alpha and beta tubulin isolated from Tetrahymena cilia. These bands were not found in the translation products of poly-A+ RNA isolated from 49-h cultures or in the translation products of poly-A- RNA.

We have analyzed the nature/content of methylated bases in the nuclear DNA of three unicellular eucaryotes. The pattern of methylation was different for each of the three organisms studied: Saccharomyces cerevisiae contained only 5-methylcytosine; Tetrahymena pyriformis contained only N6-methyladenine; and Chlamydomonas reinhardi contained both modified bases.

The effect of temperature on the core structure of endoplasmic reticulum membranes has been visualized directly in cells of the poikilothermic eukaryote Tetrahymena pyriformis by freeze-etch electron microscopy. Moreover, the effect of temperature on the smooth microsomal membrane vesicles isolated from these cells, as well as on the extracted membrane lipids, has been examined by fluorescence probing, electron spin resonance, proton nuclear magnetic resonance, and calorimetry. Freeze-etch electron microscopy of T. pyriformis cells, equilibrated at different temperatures between 28 and 5 degrees, reveals the emergence of smooth areas on the fracture faces of endoplasmic reticulum membranes at temperatures below similar to 17 degrees. In this temperature range, we also find discontinuities in the glucose 6-phosphatase activity, in the fluorescence intensity of 8-anilino-1-naphthalensulfonate, in the partition of 4-doxyldecane, and in the separation of the outer hyperfine extrema of 5-doxylstearic acid in the microsomal membranes. These membranes apparently contain at least two lipid environments of different fluidity as indicated by the 12-doxylstearic acid spin-label. Proton nuclear magnetic resonance of the extracted membrane lipids indicates an abrupt change of the fatty acid chain mobilities at temperatures below similar to 17 degrees. This, however, is not due to a true thermal liquid crystalline in equilibrium crystalline phase transition. Calorimetric measurements also support this conclusion. The thermotropic alterations observed within the membranes are interpreted to be due primarily to a clustering of "rigid" liquid crystalline lipid environments which exclude membrane-intercalating proteins.

Phosphatidylethanolamine of 15 degrees C-grown Tetrahymena pyriformis (NT-I) cells contains more polyunsaturated fatty acids than 39.5 degrees C-grown cells. This increase in unsaturation is due to an increase in linoleic (C18 : 2) and linolenic (C18 : 3) acids, and a decrease in myristic (C14 : 0), palmitic (C16 : 0), palmitoleic (C16 : 1) and heptadecanoic (C17 : 0) acids. Compared with 39.5 degrees C-grown cells, the proportion of palmitic acid (C16 : 0) decreased in the 1-position as does at the 2-position in 15 degrees C-grown cells. On the contrary, there is a significant increase in linoleic (C18 : 2 delta 9, 12) and gamma-linolenic (gamma-C18 : 3) acids in the 1- and 2-positions, respectively. Phosphatidylethanolamine has been subfractionated into seven different diglyceride species. In 15 degrees C cells, the amounts of fractions 2 (1-linolenoyl-2-linoleoyl) and 3 (1-linolenoyl-2-palmitoleoyl, 1-linolenoyl-2-oleoyl) increased while there was a great decrease in subfraction 7 (1-myristoyl-2-palmitoleoyl, 1-palmitoyl-2-palmitoleoyl). Since subfractions 1 and 2 contain over 70% linoleic (C18 : 2) and linolenic (C18 : 3) acids, these fractions might be composed mainly of 1-linolenoyl-2-linolenoyl and 1-linolenoyl-2-linoleoyl molecular species at 15 degrees C. These data support evidence that phosphatidylethanolamine would play a principal role as an acceptor of acyl chains for temperature acclimation.

A novel Tetrahymena sp. is similar in appearance to four classical strains (WH-14, GL, W, NT-1) of T. pyriformis except for a larger width. The phospholipid and acyl chain compositions of membranes of this novel species are significantly different from those of other strains. The Tetrahymena sp. has a much higher content of ethanolamine phosphoglyceride and a lower level of glyceroaminoethyl phosphonate. Major fatty acids of this strain are iso 15:0 and iso 17:1, which are, if present, usually minor components in the classical strains of T. pyriformis.

1. Two sphingophosphonolipids were isolated from the lipids of the ciliated protozoan, Tetrahymena pyriformis WH-14. They were ceramide N-methyl-2-aminoethylphosphonate (CMAEP) and ceramide 2-aminoethylphosphonate (CAEP), in yields of 0.05 mg/g and 1.74 mg/g dry cells, respectively. 2. Two chromatographically distinguishable CAEP species were found, a slow-moving major component and a minor component which moved faster; the slow-moving one contained primarily hydroxy fatty acids, while in the other one nonhydroxy fatty acids were predominant. However, their long-chain base constituents were similar. 3. The major fatty acids of CAEP were 2-hydroxy acids with carbon numbers of 16 to 19, which were almost exclusively iso-types. The fatty acids of CMAEP consisted mainly of palmitic, iso-octadecanoic, and 2-hydroxy iso-heptadecanoic acids. 4. The long-chain bases were dominated by C16, C17, and C19 iso-4-sphingenine homologs.

Ca-dependent affinity chromatography on phenothiazine-Sepharose 4B has been used to isolate a pure protein from the ciliate Tetrahymena pyriformis. This protein has been identified as calmodulin by demonstrating three of the Ca-dependent activities attributed to calmodulins. Tetrahymena calmodulin also has physicochemical properties similar to those of the previously characterized mammalian, coelenterate, and plant proteins, except for a lower molecular weight (15,000) and slightly different CNBr fragments compared to bovine brain calmodulin. Calmodulin is a constituent of demembranated Tetrahymena cilia from which it can be extracted with the crude dynein fraction. Sucrose density gradient fractionation indicated its presence in fractions containing the 14S dynein ATPase. It is concluded that the essential properties of calmodulin have been highly conserved during much of eukaryotic evolution, and it is suggested that calmodulin plays a role in the control of ciliary motility in Tetrahymena.

Three proteins (A, B and C) that bind specifically to single-stranded DNA have been isolated from the eukaryotic organism Tetrahymena pyriformis. Their molecular weights are 47 000, 41 000 and 32 000. The amino acid composition of the A protein indicates that it is a non-histone protein and sucrose gradient centrifugation shows that it binds to bacteriophage M13 DNA and to oligo (dT)100 in a cooperative manner. The exonucleolytic degradation of oligo (dT)100 is prevented when it is bound to the A protein. The effect of A protein on the exonucleolytic reaction confirms the cooperative manner of binding of A protein binding to oligo (dT)100 and shows that this process may be prevented by high ionic strength. The A protein seems to be without enzymatic activity.

Pellicles of the ciliate Tetrahymena pyriformis strain GL (phenoset A) were isolated by a new procedure. Oral apparatuses also purified by a modification of a pervious method. Both preparations were characterized by electron microsocpy. Proteins of the isolates were separated by analytical SDD 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.

Two dimensional (2D) urea-polyacrylamide gel electrophoresis of tRNA isolated from Tetrahymena mitochondria separated at least 36 spots, while more than 45 major and minor spots were resolved with cytosolic tRNA. Co-electrophoresis of mitochondrial and cytosolic tRNAs revealed that many spots co-migrate. When radioactive mitochondrial tRNA was hybridized to mtDNA under various conditions and tRNA melted from the hybrid was analyzed by 2D gel electrophoresis, only 10 tRNA spots were found. Identified as mtDNA-encoded were 2 spots for tRNA(leu), 2 for tRNA(met), and 1 each for tRNA(phe), tRNA(trp) and tRNA(tyr). The remaining three were unidentified. Mitochondrial tRNA spots that correspond to the tRNAs for arg, gly, ile, lys, ser, and val do not hybridize with mtDNA, and in gel positions they correspond to the cytoplasmic tRNA spots for the same respective amino acids. These mitochondrial tRNAs isolated from the gel can be acylated either by the mitochondrial or cytosolic enzymes. Mitochondrial tRNA isolated from a Tetrahymena cell homogenate which was pretreated with RNase A and Micrococcus nuclease exhibited the same 2D gel pattern as a non-treated control. Mitochondrial tRNAs from old and young cells showed generally similar tRNA spots in 2D gels, though more variable spots were seen with old cells. 3H-labeled whole-cell tRNA added to the cell homogenate prior to the mitochondrial isolation procedure did not remain associated with the final mitochondrial tRNA preparation. The present studies also showed mitochondrial tRNAs bound to the mitochondrial 80S monosome and polysome fractions. Radioactive tRNA added to the mitochondrial lysate does not adhere to the ribosomes, suggesting that the ribosome-bound tRNAs are not contaminating cytoplasmic tRNAs. These results are generally in good agreement with our previous data showing that only a small number of tRNAs are coded for by the mitochondrial DNA, while the others are a selected set of imported cytoplasmic tRNAs.

By phenol extraction and DEAE cellulose column chromatography, tRNA was isolated from Tetrahymena pyriformis strain GL. Following acid hydrolysis of the tRNA, the methylated purine content was determined by Dowex 50 column chromatography and paper chromatography. The most abundant methylated guanine derivative was found to be N2-DMG. Also present were 1-MG, N2-MG, and 7-MG. The most abundant methylated adenine was found to be 1-MA; no 2-MA was detected. Small amounts of the N6-methyladenines were detected.

The effect of growth of Tetrahymena pyriformis in ethidium bromide (EthBr) on the structure and synthesis of mitochondrial DNA (mtDNA) has been investigated. During the first 5 h of growth in EthBr, mtDNA synthesis is inhibited 95% or more. After 10-15 h, this block is partially released and large numbers of replicating molecules accumulate, indicating that inhibition by EthBr primarily affects the rate of chain growth and not the initiation of new rounds of replication. The accumulated molecules sediment more rapidly than normal Tetrahymena mtDNA and do not contain enough single-strand regions to distinguish them from normal Tetrahymena mtDNA when banded in buoyant CsCl or NaI gradients. Electron microscopy shows that the predominant species in this rapidly sedimenting DNA is a linear molecule containing one symmetrical double-stranded replication loop of varying size located at its center. No degradation of mtDNA from cells grown in EthBr was detected in alkaline velocity gradients.

Extracts of protozoa contain materials that resemble guinea pig insulin, which is noted for its unusual structure and properties. The protozoan derived materials react in the radioimmunoassay for guinea pig insulin; some but not all of these immunoreactive materials migrate on gel filtration in the position of authentic guinea pig insulin. Experiments were done to exclude artifacts in the assay as well as inadvertent contamination by guinea pig insulin. By immunological methods, we segregated the guinea pig type immunoactivity from that which has rat/pork type immunoactivity. These findings extend our studies of extracts of guinea pig tissues which also have these two types of insulin immunoactivities.

Poly(A)-containing RNAs with sedimentation coefficients in the range 10-45 S and 10-35 S (Mr 0.2-3.3 x 10(6) and 0.2-2.2 x 10(6) approximately) are present in the nucleus and cytoplasm of T. pyriformis respectively. The poly(A) segments present in both nuclear and cytoplasmic poly(A)-containing RNAs have a chain length of 80 - 150 nucleotides. Significant amounts of high-molecular-weight poly(A)-containing RNAs (35 S, Mr 2.2 x 10(6) are found in the post-polysomal fraction of the cytoplasm (messenger ribonucleoprotein particles?) and also in large polysomes. In order to exclude the possibility that these high-molecular-weight species are products of non-specific aggregation, RNA preparations were submitted to severe denaturing conditions before analysis and all electrophoreses were carried out in the presence of 95% (v/v) formamide.

Temperature-responsive microsomes of the ciliate protozoan Tetrahymena have been originally fractionated by step centrifugation on two-layered, Mg2+-containing sucrose gradients. Three fractions have been obtained, which are termed smooth I, smooth II and rough according to the appearance of the membrane vesicles upon electron-microscopy. Smooth I, smooth II, and rough microsomes exhibit RNA/protein ratios of 0.09, 0.20, and 0.34; their phospholipid/protein ratios and their neutral lipid/phospholipid ratios were 0.52, 0.43 and 0.25, and 0.17, 0.18 and 0.13, respectively. All three fractions contain equivalent, low succinic dehydrogenase and 5'-nucleotidase activities. Glucose-6-phosphatase and acid phosphatase are more concentrated in smooth I membranes than in rough membranes. The reverse is true for ATPase. The smooth II membranes occupy an intermediate position except that their ATPase activity is the lowest of the three fractions. The specific activities of these enzymes of the three microsomal fractions are compared to those of homogenates of whole cells. Thin-layer chromatography reveals a very similar polar and nonpolar lipid pattern of the three microsomal fractions. The major phospholipid compounds are phosphatidlethanolamine, glycerideaminoethylphosphonate and phosphatidylcholine, while diglycerides, an unknown NL-compound, and triglycerides are the major apolar lipids. Gas liquid chromatography shows that the fatty acids are mainly even-numbered ranging between C12 and C18. The smooth I, smooth II and rough membranes contain 65.2, 69.3 and 72.7% unsaturated fatty acids in their polar lipids, whereas only 52.7, 49.7 and 48.3% unsaturated acids are found in their apolar lipids, respectively. The fatty acids are more unevenly distributed among the individual polar lipids than in the apolar ones.

The H4 histone of the protozoan Tetrahymena pyriformis, as obtained previously (Nomoto, M. & Iwai, K. (1982) J. Biochem. 91, 719-723), was completely sequenced; the total sequence reported preliminarily (Hayashi, H., Nomoto, M., & Iwai, K. (1980) Proc. Jpn. Acad. 56B, 579-584) is one of the two variant sequences determined here. The intact H4 was directly sequenced by automated Edman degradation from the N-terminal through residue 92. Sequence determination was further performed with tryptic peptides and peptic peptides, both covering the whole sequences. Thus, the complete sequences of two variants were determined; both consist of a total of 102 amino acid residues, have identical compositions, have the same molecular weights of 11,228 in the unmodified form, and are partially acetylated at four lysine residues from the N-terminal. The sequences differ in two positions from each other (-Lys-Arg-/-Arg-Lys- at residues 19 and 20, 7 : 3 mol/mol), and in 22 or 20 positions from the human spleen H4 sequence. The implications of these results for the structure-function relationship of this histone species and also for the phylogeny of protozoa are discussed.

In the present communication a characterization of the 5 S rRNA genes and the tRNA genes of Tetrahymena pyriformis has been performed. The number of 5 S rRNA and tRNA genes in the macromolecular DNA has been established. Furthermore no sequence homology is observed for these genes. The number of both types of genes does not change significantly under starvation conditions. The genomic organization of the 5 S rRNA and tRNA genes has been investigated. From in vivo replication studies it is concluded, that replication of both 5 S rRNA and tRNA genes takes place throughout the whole S-period.

The addition of ergosterol to cultures of Tetrahymena pyriformis results in (a) the accumulation of the sterol by the cells; (b) the inhibition of the synthesis of the pentacyclic triterpenoid alcohol, tetrahymanol; (c) the replacement of tetrahymanol by ergosterol in the ciliate membranes. The dry weight and lipid content of sterol-supplemented ciliates did not differ from the controls. Examination of the lipid classes revealed no change in composition except for a higher content of ergosterol in supplemented cells than tetrahymanol in control cultures. The relative proportions of triglycerides, the major classes of polar lipids, 1-alkyl phospholipids and phosphonolipids, appeared unaltered. A complex array of fatty acids is found in this ciliate. Several acids not reported previously in this organism were isolated and identified, and the novel fatty acid 18:2 delta 6, 11, was found in substantial amounts. Ergosterol supplementation altered the proportions of the fatty acids, although not all lipid classes were affected to the same extent. The changes noted were of three general types: (a) a shortening of the fatty acyl chain length in the acids of the normal series; (b) a lowering in the degree of unsaturation; (c) a discrimination between two isomers of lionoleate, 18:2 delta 6, 11 and 18:2 delta 9, 12. The former is elevated in the presence of ergosterol while the latter is depressed. Each class of polar lipids has a distinctive fatty acid composition. Among the glycerophospholipids, cardiolipin and phosphatidylcholine were least affected, while the mixture of 1-alkyl-2-acyl-sn-glycero-3-(2-aminoethyl)-phosphonate and 1,2-diacyl-sn-glycero-3-(2-aminoethyl)-phosphonate was most markedly altered. Sphingolipid fatty acid composition was influenced by ergosterol supplementation. Two changes were noted: (a) a reduction in the length of the hydrocarbon chain; (b) an increase in the proportion of alpha-hydroxy acids. The impact of ergosterol on the fatty acid composition of the polar lipids may be on fatty acid biosynthesis, on incorporation of fatty acids, or on the turnover rates of the fatty acyl groups. Ergosterol is concentrated in the ciliary (limiting) membrane, as are the polar lipids most affected. This localization allows the speculation that the change in fatty acid composition may be related to the maintenance of optimal membrane properties upon the introduction of the sterol.

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 approximately 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.

Isolated microtubule protein from axopodia of the heliozoan Echinosphaerium nucleofilum, consisting of two major bands on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), has been compared to axonemal and cytoplasmic tubulins from both animal and non-animal sources. The upper E. nucleofilum protein band migrated faster than the alpha-tubulins of bovine brain and sea anemone sperm tails but with approximately the same electrophoretic mobility as the axonemal alpha-tubulins of Tetrahymena pyriformis and the alga Chlorogonium elongatum and cytoplasmic alpha-tubulin from the slime mold Physarum polycephalum. The lower E. nucleofilum protein band, however, had a higher electrophoretic mobility than all the beta-tubulins which we have so far examined. It was, nevertheless, a true beta-tubulin as shown by its migration on two-dimensional gel electrophoresis and the general resemblance of its one- and two-dimensional peptide maps to those of other beta-tubulins. The Staphylococcus aureus protease cleavage pattern of the upper axopodial protein band was similar to those of other non-animal alpha-tubulins but quite different from those of the animal alpha-tubulins. In contrast, the two-dimensional tryptic peptide map of axopodial alpha-tubulin was distinct from all of them. For example, a characteristic constellation of peptides common to the peptide maps of the other alpha-tubulins was absent from that of E. nucleofilum. In contrast to Physarum and metazoan tubulins but similar to Tetrahymena tubulin, the axopodial alpha-tubulin had a more basic isoelectric point than the beta-subunit as shown by two dimensional gel electrophoresis. Some of the unusual characteristics of E. nucleofilum axopodial tubulin may not only reflect phylogenetic variation, but also the different functional requirements of axopodial microtubules.

Isolation and characterization of a fibrous protein component which might be associated with contractile ring of a dividing Tetrahymena cell were attempted by making use of coprecipitation of the protein with rabbit skeletal muscle myosin. The protein was purified to homogeneity by ammonium sulfate fractionation between 40--70% saturation and column chromatography of Sephadex G-200, starting from KCl-extract of Tetrahymena acetone powder. Its molecular weight was calculated to be 38,000, based on the electrophoretic mobility in sodium dodecyl sulfate (SDS)-polyacrylamide gel, whereas molecular weight of its native state was determined to be 140,000 by gel filtration on Sephadex G-200, and its sedimentation coefficient was about 9S as estimated by sucrose density gradient centrifugation. The latter was a particle of 7.7 nm in diameter under an electron microscope and supposed to be a tetramer of the 38,000-dalton protein. The protein is considered to be a new, unique protein, since it is definitely different from the ubiquitous non-muscle actin in molecular weight, polymerizability in KCl solution and amino acid composition, and it also different from tropomyosin and tubulin in immunological characteristics and amino acid composition.

The large amounts of dopamine accumulated by cells of Tetrahymena pyriformis strain NT-1 and secreted into their growth medium were found to depend primarily upon an extracellular, non-enzymatic conversion of tyrosine to L-dihydroxyphenylalanine (L-DOPA); L-DOPA was then rapidly taken into the cells and transformed into dopamine enzymatically. Efforts to find physiologically significant dopamine binding sites on the cell surface or dopamine-sensitive adenylate cyclase activity were unsuccessful, suggesting that the catecholamine does not function in Tetrahymena as it does in higher animals.

Examinations with fluorescein isothiocyanate (FITC)-bovine serum albumin histamine conjugate and with FITC-labeled antibodies to histamine have shown that the unicellular Tetrahymena possess histamine binding sites (receptors) which are localized on the cilia of the cell body. No histamine is bound by the interciliar membrane regions and by the cilia of the oral fields.

Lead acetate (0.1--0.2%) forms a precipitate with the organic growth medium. The Tetrahymena cells ingest this lead-containing precipitate and cell growth is resumed after a variable lag period. Ingested lead is observed as electron-dense material in food vacuoles. Soon after exposure, cytoplasmic lead (preserved with certain fixation only) is revealed as electron-dense particles in cilia and in a halo around digestive vacuoles. Later the lead particles pervade the entire cell but after the lag period they are confined to membrane-bound spaces. In dilute growth medium, high concentrations of lead inhibit food-vacuole formation and cell growth. Under these conditions lead is deposited in alveoli of the pellicle and is also found in autophagic vacuoles and other membrane-limited structures. The study has revealed that lead enters Tetrahymena through the membrane of digestive vacuoles and through the cell surface. The change in distribution of lead during the lag period indicates that a mechanism is activated for removal of lead into membrane-bound spaces. The final storage of lead seems to be in lysosomes.

A method is described that enables a chromatin fraction containing ribosomal DNA (DNA containing sequences coding for rRNA) to be prepared from the macronuclei of growing or stationary cultures of Tetrahymena pyriformis. This material is obtained in yields of between 25 and 75% of the theoretical maximum. The DNA in this fraction was identified as ribosomal DNA on the basis of its density and molecular weight, and it appears not to be appreciably contaminated by other DNA. The method relies on the approximate assumption that ribosomal DNA is the smallest species of DNA in chromatin in the nucleus, and avoids the use of mechanical force, or enzyme action, to fractionate chromatin.