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Okadaic acid, a specific inhibitor of protein phosphatase 1 in Paramecium causes sustained backward swimming in response to depolarising stimuli (S. Klumpp et al. (1990) EMBO J. 9, 685). Here, we employ okadaic acid, tautomycin, microcystin LR and inhibitor 1 as phosphatase inhibitors to identify a 42 kDa protein in the excitable ciliary membrane that is dephosphorylated by protein phosphatase 1. Identification of the 42 kDa protein was facilitated by the finding that the protein kinase responsible for its phosphorylation uses Ca-ATP as a substrate just as effectively as Mg-ATP. Notably, dephosphorylation of the 42 kDa protein is specifically inhibited by cyclic AMP; cyclic GMP has no effect.

A class of Paramecium behavioral mutants called pantophobiacs have a deficiency in calcium-dependent potassium efflux, and this deficiency can be corrected by the microinjection of wild-type Paramecium calmodulin (Hinrichsen, R. D., Burgess-Cassler, A., Soltvelt, B. C., Hennessey, T., and Kung, C. (1986) Science 232, 503-506). As a starting point in investigations of which features allow wild-type Paramecium calmodulin to fully restore this behavior while other calmodulins are inactive or poorly effective, we elucidated the amino acid sequence of the wild-type calmodulin. We utilized an approach that combined Edman chemistry with mass spectrometry. This approach resulted in the identification of a new post-translational modification in calmodulin: N epsilon,N epsilon-dimethyllysine at residue 13. This particular modification has not been described for calmodulins studied previously. The only other first-domain modification that has been described for any calmodulin is acetylation of the amino terminus (Watterson, D. M., Sharief, F., and Vanaman, T. C. (1980) J. Biol. Chem. 255, 962-975). These results along with analyses of pantophobiac calmodulin and calmodulin binding proteins will provide insight into calmodulin's role in a well-defined behavioral mutant.

The macronuclear ribosomal DNA (rDNA) of the ciliated protozoan Paramecium tetraurelia (stock 51) was analyzed by digestion with restriction endonucleases. The fragments which contained ribosomal RNA (rRNA) coding sequences and spacer sequences were identified. The spacer sequences exhibited some heterogeneity in size. The genes coding for 5.8S RNA, but not for 5S RNA, are linked to the 17S and 25S rRNA genes. Complementary RNA, synthesized from rDNA of stock 51, was hybridized with restriction digests of whole cell DNA from six other allopatric stocks of this species. The restriction patterns of the rDNA from these seven stocks were, in general, very similar, and the sizes of the coding sequences were identical in all seven stocks. Only the restriction pattern of rDNA from stock 127 differed significantly from that of stock 51. The rDNA from stock 127 was isolated and characterized, and with the exception of the restriction pattern of its spacer, it resembled the rDNA from stock 51. It is concluded that the rDNA repeat in Paramecium, including the spacer, has, in general, been conserved during the course of evolution. It is suggested that in some species, even in the absence of genetic exchange among geographically separated populations, selection pressure may act to conserve spacers of tandemly repeated rDNA. The conservation may be related to the number of rDNA copies in the germinal nucleus.

Fatty acid positional distributions and the fatty acid compositions of diacyl and alkyl acyl analogs of the three major glycerophospholipids of Paramecium tetraurelia were examined. Phosphatidylcholine and the phospho- and phosphonyl ethanolamine glycerolipids were separated into diacyl and alkyl acyl species by thin-layer chromatography after phospholipase C digestion, and acetylation. Phosphatidylcholine and the ethanolamine phosphonolipid were predominantly in the alkyl acyl form and phosphatidylethanolamine was predominantly in the diacyl form. The three major glycerophospholipids were also subjected to hydrolysis by phospholipase A2. Complete and efficient hydrolyses of all three phospholipids were accomplished with the enzyme from porcine pancreas. Sodium tetraborate prevented acyl migration when added to reaction mixtures. Fatty acids released by phospholipase A2 action, and the lysoderivatives were separated by thin-layer chromatography. Fatty acid methyl esters were prepared and analyzed by gas-liquid chromatography. Saturated acids were predominantly at C-1 of diacyl phospholipids. Polyunsaturated fatty acids were mainly at C-2, particularly in the alkyl acyl species. An exception was gamma-linolenate which was a major acid found esterified to C-1 in the three diacyl phospholipids. Identification of this acid at that position supports the idea that in some ciliates there may be an acyltransferase, specific for gamma-linolenate, that esterifies this acid to the glycerophospholipids.

As a first step in the biochemical analysis of membrane excitation in wild-type Paramecium and its behavioral mutants we have defined the protein composition of the ciliary membrane of wild-type cells. The techniques for the isolation of cilia and ciliary membrane vesicles were refined. Membranes of high purity and integrity were obtained without the use of detergents. The fractions were characterized by electron microscopy, and the proteins of whole cilia, axonemes, and ciliary membrane vesicles were resolved by SDS polyacrylamide gel electrophoresis and isoelectric focusing in one and two dimensions. Protein patterns and EM appearance of the fractions were highly reproducible. Over 200 polypeptides were present in isolated cilia, most of which were recovered in the axonemal fraction. Trichocysts, which were sometimes present as a minor contaminant in ciliary preparations, were composed of a very distinct set of over 30 polypeptides of mol wt 11,000--19,000. Membrane vesicles contained up to 70 polypeptides of mol wt 15,000--250,000. The major vesicle species were a high molecular weight protein (the "immobilization antigen") and a group of acidic proteins with mol wt similar to or approximately 40,000. These and several other membrane proteins were specifically decreased or totally absent in the axoneme fraction. Tubulin, the major axonemal species, occurred only in trace amounts in isolated vesicles; the same was true for Tetrahymena ciliary membranes prepared by the methods described in this paper. A protein of mol wt 31,000, pI 6.8, was virtually absent in vesicles prepared from cells in exponential growth phase, but became prominent early in stationary phase in good correlation with cellular mating reactivity. This detailed characterization will provide the basis for comparison of the ciliary proteins of wild-type and behavioral mutants and for analysis of topography and function of membrane proteins. It will also be useful in future studies of trichocysts and mating reactions.

In this paper we demonstrate the presence and localization of calmodulin, a calcium-dependent regulatory protein, in the ciliated protozoan Paramecium tetraurelia. Calmodulin is demonstrated by several criteria: (a) the ability of whole cell Paramecium extracts to stimulate mammalian phosphodiesterase activity, (b) the presence of an acidic, thermostable, 17,000-dalton polypeptide whose mobility shifts in SDS polyacrylamide gel electrophoresis in the presence of Ca2+, and (c) the affinity of antibodies against mammalian calmodulin for a Paramecium component as demonstrated by both indirect immunofluorescent localization and radioimmunoassay. Indirect immunofluorescence studies reveal that Paramecium calmodulin is distributed in three distinct regions of the cell, i.e., (a) large, spherical cytoplasmic organelles representing perhaps the food vacuoles or other vacuolar inclusions of the cell, (b) along the entire length of oral and somatic cilia, and (c) along a linear punctate pattern corresponding to the kinetics (basal bodies) of the cell.

In vivo labeling of Paramecium cells with 32Pi most heavily labels a minor 63-kDa protein that undergoes a rapid, Ca2+-dependent dephosphorylation when the cell is stimulated to release. This stimulus-sensitive phosphoprotein was isolated and purified to apparent homogeneity. A polyclonal affinity purified antibody made against the purified protein recognizes both the phosphorylated and dephosphorylated forms of the protein. The phosphorylated 63-kDa protein is found in the cytosolic fraction; it is slightly acidic with two isoelectric forms at pI 5.8 and 6.2 and probably exists as a monomeric 60-65-kDa polypeptide in the native state. The labeled phosphoamino acid of the protein is phosphoserine. The affinity purified antibody recognizes a third isoelectric form at pI 6.3 that appears unlabeled. The specificity of the antibody was confirmed by showing that it immunoprecipitates the correct protein, i.e. the stimulus-sensitive 63-kDa phosphoprotein. The availability of purified 63-kDa protein as well as an antibody against it will now allow molecular, biochemical, and immunocytochemical studies into the role of this protein in the mechanism of exocytosis.

The secretory contents ("matrix") of Paramecium tetraurelia trichocysts expand by a factor of 4.5 when they undergo a Ca2+-mediated decondensation in the course of exocytosis. This is paralleled by a concomitant increase in the interval of the periodic banding of the matrix from 12 nm to 45-51 nm, which becomes visible with different electron stains for proteins and negatively charged groups. Recent reports of actin in secretory contents led us to investigate its redistribution and artifactual adsorption to the trichocyst contents upon their expansion. To visualize this effect we used peroxidase-labeled F(ab) fragments from an IgG directed against Paramecium actin, a DNAase I-gold complex, and the induction of F-actin polymerization. The trichocysts were analyzed in situ as well as after isolation by density-gradient centrifugation. Additionally, in response to current reports in the literature, we reanalyzed trichocyst contents for any possible presence of calmodulin. We applied three independent in situ methods for this: autofluorescence after trifluoperazine affinity labeling, calmodulin-fluorescence affinity labeling, and an electron microscopic immunocytochemical method. All three methods failed to reveal any significant labeling of structurally intact trichocysts in situ, although we also showed that discharged trichocysts avidly adsorb calmodulin from the culture medium. From the present data we conclude that the decondensation of trichocysts during exocytosis is mediated by a sudden conformational rearrangement of secretory proteins in the trichocyst contents, without the involvement of any other regulatory or contractile proteins, which occur only in the cytoplasm. Trichocyst contents are not significantly--if at all--glycosylated.

Using a monoclonal antibody generated against striated muscle troponin T, we previously noted the presence of crossreactive components in smooth muscle and non-muscle cells. Since the presence of troponin T in tissues other than striated muscle is controversial, we sought to establish the nature of the crossreaction and to determine the extent of its occurrence. For this study, indirect immunofluorescence microscopy and immunoblot analyses were performed. Crossreactive material was found in diverse cells from the animal, plant and fungal kingdoms. On the basis of morphological distributions, both microtubule-associated and non-microtubule-associated components were revealed. Microtubule-associated components of animal cell lines included a 35 X 10(3) Mr protein, similar in electrophoretic mobility to skeletal troponin T (37 X 10(3) Mr). Reactive components of comparable mobility were observed in immunoblots of brain and cerebellar homogenates. Filamentous staining was observed in a variety of mammalian cells in culture and in cells of vertebrate tissues. Chick cerebellar tissue showed reactions in the neurites of the molecular layer and granule cell bodies. In the plant kingdom, examination of the onion root-tip cells indicated an association of crossreactive components with interphase cortical microtubules, preprophase bands, the mitotic spindle and phragmoplast microtubules. In the fungal kingdom, both interphase and mitotic spindle microtubules in a cellular slime mould were reactive. Non-microtubule-associated components were observed in the centrosphere regions of mitotic sea-urchin eggs, in mitotic and interphase plasmodia of Physarum polycephalum, and in trichocysts and basal bodies of Paramecium tetraurelia. In all systems examined, the troponin T crossreactive components were located in regions or on structures of possible Ca2+ or calmodulin activity, suggesting a possible functional similarity to troponin T.

Six ethanolamine sphingophospholipids from axenically cultured Paramecium tetraurelia were isolated from cells and purified ciliary fractions, and were characterized. The sphingolipids comprised 10.7% of whole cell and 32.5% of ciliary ethanolamine phospholipid fractions purified by ion exchange column chromatography. The individual sphingolipids were characterized by thin-layer chromatographic analyses of parent compounds and the polar head group and long chain base moieties, gas-liquid chromatography, and mass spectrometry of amide-linked fatty acids and long chain bases, and nuclear magnetic resonance of the compounds. Colorimetric assays of differential hydrolysis products and 31P nuclear magnetic resonance were used to determine the nature of phosphorus linkages. The sphingolipids were identified as N-acyl-trans-4-hydroxy-sphinganine-1- phosphonoethanolamine , N-acyl-trans-4-hydroxy-sphinganine-1-phosphoethanolamine, N-acyl-sphingenine-1- phosphonoethanolamine , N-acyl-sphingenine-1-phosphoethanolamine, N-acyl-sphinganine-1- phosphonoethanolamine and N-acyl-sphinganine-1-phosphoethanolamine. All six had greater than 90% saturated fatty acids. These sphingolipids were quantified by radioisotope methods and plate densitometry of thin-layer chromatograms. Changes in the relative amounts of each species were detected in cells grown in different culture media as well as in cells at different culture ages.

Procedures and instrumentation are described to extend the capability of a cytometry system to record samples that exhibit a wide range of fluorescence such as multicellular systems. The method employs a log amplifier in combination with a set of neutral density filters that reduces the incident light reaching the photomultiplier tube. With any given filter, signals within an intensity range of 200-fold can be measured; different filters can be used to obtain an extended overall range. Polystyrene fluorescent microspheres and a variety of mithramycin stained biological samples ranging from yeast cells to Paramecium were processed by the system. The relative DNA content of individual multicellular embryos was determined for a heterogeneous population of embryonic stages isolated from the nematode, Caenorhabditis elegans. As part of the evaluation of the procedure, the practical upper limit of range extension was determined. The most intense fluorescent signal was produced when untreated pecan pollen stained with ethidium bromide fluoresced with a factor (8.4 +/- 1.3) X 10(4) more than ethidium bromide stained E. coli cells.

Macronuclear DNA was isolated from purified macronuclei of Paramecium aurelia and the size distribution was determined with regard to growth phase and method of extraction. DNA molecules as long as 105 microns and as short as 0.2 microns were observed. It was concluded that the method of extraction affected the observed length of DNA extracted and that macronuclear DNA isolated from cells in balanced growth was less susceptible to nuclease degradation than was DNA isolated from cells in stationary phase. Renaturation studies were performed on macronuclear DNA and a kinetic complexity of 22-times E. coli DNA was determined. This value was similar to those values reported for Tetrahymena and Stylonychia macronuclear DNA. Correcting for GC base content yielded a kinetic complexity for Paramecium macronuclear DNA of 11-times E. coli DNA which corresponded to 3 X 10(10) daltons. There would be about 1400 copies of a unit genome of this complexity within each newly replicated macronucleus. Density gradient analysis indicated that the genes coding for ribosomal RNA had a greater density in CsCl than the bulk DNA. Molecular hybridization studies indicated that the genes coding for 25 S RNA represented 0.14 percent of the total macronuclear DNA. Correcting for GC base content, this corresponded to 30-35 25 S RNA genes per unit genome. These results on Paramecium are discussed in relationship to other ciliate macronuclear DNA.

Several important points of heme-peptide cytochemistry were quantitatively analyzed, with particular regard to their use in electron microscopic immunocytochemistry. A simple procedure is presented for the preparation of heme-octapeptide (H-8-P) microperoxidase. H-8-P, hemenonapeptide (H-9-P), and various horseradish peroxidase (HRP) isoenzymes were used for coupling with immunoglobulin (Ig)G or the papain-cleavage fragments from IgG (Fab) molecules. Ultracentrifugation and spectrophotometric analyses revealed the following characteristics of the conjugates: a) They are of a uniform size class; b) their diameters were calculated, and ranged from 5.6 (Fab-H-8-P; H-9-P) to 10.5 (IgG-HRP); c) the persistence of antigen binding capacity was ascertained; d) the deactivation of the marker peroxidase activity due to coupling was as low as 20-30%; e) optimal conditions for use of the electron microscopic (EM) with 3,3'-diaminobenzidine media were elaborated (with a pH optima somewhat different from some standard methods in current use); and f) on the basis of the quantitative data presented, an optimal compromise (either in favor of higher peroxidase activity with HRP conjugates or of smaller size with microperoxidase-Fab conjugates) can be achieved. Finally, the identification of isolated purified actin and of actin in cortical microfilament bundles and ciliary basal bodies of Paramecium cells served as a test object for the usefulness of conjugation products and optimized assay conditions for EM immunocytochemistry.

The synthesis of a surface protein has been studied in Paramecium through double-labeling experiments using [(14)C]- and [(3)H]leucine-labeled bacteria as the source of radioactive amino acid. Over a 4-5 h incubation period, the turnover rate was found to be higher than that of overall cell protein. In addition, the initial label is apparently utilized during the chase period, being incorporated into protein via a puromycin insensitive pathway.