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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.

A diacyltaurolipid was found in cells of Tetrahymena mimbres (formerly Tetrahymenas pyriformis NT-1). The lipid accounted for about 0.05% of the total lipids of the cells, and was composed of taurine (1 equiv.), 3,7,13-trihydroxystearic acid (1 equiv.) and non-hydroxy fatty acids (2 equiv.). By mild alkaline hydrolysis, non-hydroxy fatty acids (2 equiv.) and 2-(3,7,13-trihydroxyoctadecanoyl)aminoethanesulfonic acid were obtained. For determination of the positions of the ester bonds of the lipid, the free hydroxy group of the intact lipid was converted to a ketone, and the esterified hydroxy groups were acetylated after methanolic hydrochloric acid hydrolysis. By mass and nuclear magnetic resonance spectrometry, the structure of the derivative was identified as methyl 3,7-diacetoxy-13-oxooctadecanoate. From these results, the diacyltaurolipid isolated from T. mimbres was identified as 2-(3,7-diacyloxy-13-hydroxyoctadecanoyl)aminoethanesulfonic acid (7-acyltaurolipid A). This structure suggests that the diacyltaurolipid may be formed by acylation of taurolipid A.

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 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.

Assays were performed to determine the presence of serotonin (5-hydroxytryptamine) in Tetrahymena pyriformis GL in two different media using two different assay procedures. Serotonin was present in quantities consistent with those observed in other varieties of protozoa. The serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) was also present, suggesting that the enzyme, monoamine oxidase (MAO), provides for serotonin deamination. Inhibition of MAO in Tetrahymena was apparent due to resulting accumulation of serotonin and decreased levels of 5-HIAA. The data suggest that serotonin mediates a functional role in Tetrahymena pyriformis GL and that its metabolism may be relevant to such functions.

We developed a simple new method for the isolation of calmodulin, a ubiquitous Ca2+-binding protein, by using tubulin-Sepharose 4B column chromatography, and succeeded in rapid isolation of calmodulin from Tetrahymena pyriformis. The procedure was also shown to be successfully applicable to the isolation of calmodulins from starfish ovary, porcine brain, and monkey brain and, therefore, may be of general use for the rapid isolation of calmodulin.

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.

Dolichols of Tetrahymena pyriformis were isolated and characterized by TLC, HPLC and mass spectrometry. Four strains of Tetrahymena were studied and found to have relatively small amounts of dolichol, from 0.26 to 2.60 mg dolichol/kg wet weight. All four strains had approximately the same relative proportions of isoprenologs, dolichol-13 (2%), dolichol-14 (74%), dolichol-15 (23%), and dolichol-16 (less than 1%). Tetrahymena dolichols were found mainly in the mitochondrial subcellular fraction (86%). The pellicle fraction contained 9% and the microsomal fraction, 5% of the remaining dolichol. Free dolichol has also been found in the mitochondrial fraction of four other organisms. We were not able to demonstrate dolichyl esters in these organisms, but their presence is inferred, because reduced yields of dolichol were obtained if the lipid extracts were not saponified prior to HPLC assay.

Histone fractions have been isolated from the macronucleus of Tetrahymena pyriformis. Five classes of macronuclear histone were purified, using a combination of gel exclusion and ion-exchange chromatography, and were examined with respect to their solubility, electrophoretic, chromatographic, and chemical properties. Tetrahymena H4 is very similar to vertebrate H4, except that it exhibits a larger number of acetylated subfractions. In contrast, the other Tetrahymena histones vary more extensively from their calf thymus counterparts. Tetrahymena H3 resembles calf thymus H3 in its solubility properties and is the only macronuclear histone containing cysteine. However, it differs from vertebrate H3 in composition and has a faster electrophoretic mobility on both urea-acrylamide and sodium dodecyl sulfate-acrylamide gel electrophoresis. Tetrahymena H3 also displays a level of acetylation higher than that reported for its vertebrate homologue. Approximately 45% of macronuclear H2B, which resembles calf thymus H2B in composition and solubility, is present in a (mono)acetylated form, not detected in vertebrate somatic H2B. H1, though similar to its calf thymus homologue in solubility, modification (by phosphorylation), and other properties, differs considerably in its content of basic, acidic, and hydrophobic amino acids. Tetrahymena does not contain a histone strictly homologous to H2A. Although macronuclear histone X resembles H2A in chromatographic and some solubility properties more like H2B than H2A. Fraction X is polymorphic in sodium dodecyl sulfate-acrylamide gels, migrating as two distinct molecular forms. While it is possible that one form is H2A-like and the other more H2B-like, the observation that both forms of X behave identically in solubility fractionation schemes makes this unlikely. Fraction X is both phosphorylated and acetylated which, in addition to two molecular forms, results in a characteristic heterogeneous pattern on urea-acrylamide gels. Characterization of the histone complement of this lower eucaryote should contribute to the understanding of the evolution and biological role of these basic proteins. Moreover, this description represents the most extensive analysis to date of the histones associated with an amitotic, genetically active nucleus. It will serve as a reference to which the histones of the morphologically distinct, mitotically dividing, and genetically inactive micronucleus of this organism can be compared.

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.

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.

The complete amino acid sequence of Tetrahymena pyriformis H2B histone was determined. The histone was obtained as described in the preceding paper [Nomoto, M. & Iwai, K. (1982) J. Biochem. 91, 719--723]. The purified histone was digested with an arginine-specific protease, clostripain, and the peptides, fragmented at 7 arginyl bonds and also at many of the 20 lysyl bonds, were fractionated by repeating column chromatography; most of these peptides were sequenced by Edman degradation. The chymotryptic peptides overlapping the clostripain peptides were obtained by limited or more extensive digestion of intact histone. The sequencing of these peptides led to reasonable aligning of the clostripain peptides. Thus, the sequence of 119 amino acid residues (mol. wt, 13,316 for the unmodified form) has a completely alpha-N-blocked proline at residue 1 and a partially epsilon-N-acetylated lysine at residue 3. This sequence is compared with the known sequences of calf thymus and other H2B histones, and the implications for the structure and function relationship of this histone species and also for the phylogeny of protozoa are discussed.

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.

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.

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.

As a step to study the biological function of a new protein, fiber-forming protein-38,000 (FFP-38), which was previously isolated from Tetrahymena pyriformis, its intracellular localization was investigated by using antiserum specific for FFP-38. Double immunodiffusion tests revealed that the anti-FFP-38 serum reacted with the protein(s) in isolated organelles such as oral apparatus, pellicles and mitochondria, to give rise to a precipitin line confluent with the line formed between the antiserum and the FFP-38. Furthermore, indirect fluorescent antibody staining showed that fluorescence was intensely localized in the oral apparatus and faintly distributed throughout the cytoplasm. In particular, distinct fluorescence was found along the division furrow of dividing Tetrahymena.

Methods have been developed to analyze the kinetics of digestion of chromatin by nucleases. Radioactively labeled nuclei were incubated with enzyme in an ultrafiltration apparatus and digestion rates of different chromatin samples were computed employing a least-squares curve fitting technique to fit the data to zero-order and/or first-order kinetic models. These methods allow detailed kinetic analyses on small amounts of chromatin. Two biological systems were studied. 1) Tetrahymena thermophila macronuclei and micronuclei were compared; these nuclei differ in their transcriptional activities. 2) Ribosomal DNA (rDNA) of Tetrahymena pyriformis, approximately 60% of which codes for rRNA, can be preferentially labeled during starvation-refeeding; its digestion kinetics relative to bulk chromatin were studied. DNase I digested 20-40% of the macromolecular DNA about 3 times faster than bulk macronuclear or micronuclear DNA, and 60-80% of ribosomal gene-containing chromatin about 5 times faster than bulk chromatin. Filter hybridization studies of the DNAase I sensitivity of tRNA, 5S RNA, and ribosomal genes yielded similar results. These data are consistent with the observation that transcribed genes are especially sensitive to attach by DNase I and suggest that activated chromatin structure as probed by extensive DNase I digestion is the same in higher and lower eucaryotes for genes transcribed by all three RNA polymerases. Digestion kinetics of micrococcal nuclease were found to depend on the digestion conditions employed. These two biological systems and the methods we have developed should facilitate analyses of the factors responsible for maintaining an active chromatin structure.

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.

The histone compositions of a chromatin fraction containing ribosomal DNA and of the remaining macronuclear chromatin of Tetrahymena pyriformis was analysed by gel electrophoresis. These chromatin fractions were used as models for transcriptionally active and inactive chromatin respectively. The extent of histone modification, as indicated by the distribution of histone between differently charged subspecies in acid-urea gels, is not grossly different in the two chromatin fractions. However, histone H1 is present, but may be differently modified in the two chromatin fractions. The histone/DNA ratio in ribosomal chromatin, measured after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of samples of chromatin, was found to be the same whether chromatin was extracted from growing or stationary organisms, and to be approx. 40% of this ratio in the remaining macronuclear chromatin. The implications of these results for the possible structure of transcriptionally active chromatin are discussed.

Steady-state fluorescence polarization measurements of 1,6-diphenyl-1,3,5-hexatriene in microsomal lipids from Tetrahymena pyriformis cells grown at 39 or 15 degrees C revealed discrete slope discontinuities in plots of polarization vs. temperature. Two well-defined 'break points' were present in the 0-40 degrees C temperature range examined and their precise location was dependent upon the growth temperature of the cells. By mixing phospholipids from cells grown at different temperatures, the break points at 17.5 and 32 degrees C in 39 degrees C-lipid multilayer preparations were shown to correlate with the breaks at 12 and 27 degrees C, respectively, in similar preparations from 15 degrees C-grown cells. The discrete break points were also present, but at slightly different characteristic temperatures, in a phosphatidylcholine fraction and a phosphatidylethanolamine plus 2-amino-ethylphosphonolipid fraction purified from the phospholipids and in total microsomal lipids (phospholipids plus the sterol-like triterpenoid, tetrahymanol). However, catalytic hydrogenation of the phospholipid fatty acids or mixing the non-hydrogenated phospholipids with increasing proportions or synthetic dipalmitoyl phosphatidylcholine eliminated the break points. We interpret this discontinuous thermotropic response in microsomal lipids as signalling a lipid phase separation of importance in regulating physiological events.

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.

We have reported that extracts of Escherichia coli and Tetrahymena grown in synthetic media contained material that reacted specifically in the immunoassay and bioassay for insulin. One additional strain of Tetrahymena and four of E. coli yielded amounts of material similar to those reported previously. In addition to their behavior on Sephadex G-50, the immunoactive insulin-related materials from the microbial sources behaved like authentic vertebrate insulins in their ability to be adsorbed to and eluted from disposable octadecasilylsilica cartridges, DEAE-Sephadex, DEAE-cellulose, and one system of high-pressure liquid chromatography (HPLC). As with less purified microbial material, the "insulin" that had been purified on DEAE and HPLC, when tested for its bioactivity, had an immunoactivity:bioactivity ratio of approximately unity and the bioactivity was largely neutralized by anti-insulin antibody. Because the material from the microbes was so similar to authentic insulins, studies were undertaken to demonstrate that inadvertent contamination with vertebrate insulins was highly unlikely. Blanks carried through the entire procedure were always negative. Tetrahymena grown and extracted in another laboratory gave the same results. Tetrahymena that had been grown but then allowed to stand in the fermenter under adverse conditions and then carried through the entire procedure were devoid of insulin. Tetrahymena that were homogenized and subjected to acid hydrolysis were devoid of insulin. Further substantiation that exogenous contamination was highly unlikely was provided by two other types of experiments. In one of these, it was shown that the subcellular distribution of exogenously added porcine insulin or porcine 125I-labeled insulin was different from the distribution of endogenous insulin. In the second type of experiment, it was shown that during the log phase of growth of Tetrahymena or of E. coli the insulin content of the system increased multifold in a fashion that might be expected for living organisms but quite unexpected for exogenous contamination. (Interestingly, the insulin content of the E. coli medium far exceeded that which might be contributed by death of cells, estimated by the content in the medium of an intracellular enzyme.) When E. coli was grown and processed in four other laboratories having no contact with our own, similar levels of insulin-related material were recovered.

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.

The sonication of 30 S dynein obtained from Tetrahymena cilia induced dissociation into 14-S subunits, some of the enzyme still remaining as intact 30 S dynein and partially dissociated dynein (21 S) in a minor amount. It was demonstrated that the enzymatic properties of the 14 S subunit are quite similar to those of 30 S dynein except for the Ca2+:Mg2+ ratio. ATPase (EC 3.6.1.3) (ATP phosphohydrolase activity of the 14 S subunit was steadily enhanced by increasing concentrations of Mg2+. It was also activated by Ca2+ with an optimum at 6 mM but inhibited by a further increase in concentration. The Ca2+:Mg2+ ratio at 1 mM was about 0.62. 0.6 M KCl stimulated ATPase activity of the 14 S subunit two-fold. The Mg2+-ATPase had an optimum at pH 6.2 and revealed a high activity over pH 10. The Ca2+-ATPase showed two optima at pH 6.2 and 9.5. The Km for ATP was 10 muM. Only 10% of the 14 S subunit recombined with the outer fibers in the presence of Mg2+. The 14 S subunit was shown to have the same mobility as that of 30 S dynein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

1. Thymosin alpha-1, like reactivity, was found in several different species (insects, crab, protozoan, fungus and bacteria) by radioimmunoassay and immune fluorescence and as an extracellular product from the bacterial genus Mycobacterium. 2. Biochemically, thymosin alpha-1 has been isolated from combined crab visceral and nervous tissue by reverse phase HPLC. 3. The identification of thymosin alpha-1 in lower life forms suggests a more generalized exocrine origin in unicellular organisms prior to the development of the immune system or exocrine differentiation.

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.

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 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.

1. We have studied denatured Tetrahymena mtDNA by electron microscopy using the formamide technique. 2. After denaturation all DNA is single stranded, but within a few minutes single-stranded circles with a duplex tail are formed. 3. The duplex tail is 1.3 mum long, i.e. 8 percent of the length of native mtDNA, and it often contains a small single-stranded eye. 4. Digestion of the duplex DNA with exonuclease III of Escherichia coli abolishes its ability to form circles and duplex tails after denaturation. 5. Renaturation of denatured mtDNA leads to the formation of duplex circles with single-stranded section and/or duplex tails. In addition, a minority of duplex circles without apparent tails is formed, but these circles contain a small ambiguous section. 6. We conclude that this mtDNA contains a long terminal duplication-inversion, that could be involved in the replication of this linear mtDNA.

We have examined the ribosomal structural proteins isolated from vegetatively growing Tetrahymena pyriformis and from cells that had been starved of all nutrients for 24 h. Reproducible, nonartifactual differences in protein complement, primarily associated with the large ribosomal subunit, were found. The kinetics of change in ribosomal protein complement were followed both in refed and in newly starved cells. Furthermore, attempts at correlating a certain protein "phenotype" with a particular functional state of the ribosome were made. It was concluded that the alterations seen could not be correlated with a specific stage in the normal ribosome cycle. We did show, however, that the change in protein complement could occur as a result of altering preexisting ribosomes. In addition, we showed that the change correlates with a decrease in growth rate rather than being caused by the starvation conditions themselves. Speculations as to the functional significance of the protein changes are presented.