Antibodies against Tetrahymena 14-nm filament-forming protein recognize the replication band in Euplotes.

Tetrahymena 14-nm filament-forming protein (49K protein) is a structural protein which is involved in activity of the pronuclei during conjugation (O. Numata, T. Sugai, and Y. Watanabe (1985) Nature (London) 314, 192-194). Using monoclonal and polyclonal antibodies, we here demonstrate the presence of a cross-reactive protein (CRP-49) within the macronuclear replication bands of Euplotes harpa and E. eurystomus which is recognized by anti-49K protein antibodies. Immunoblotting reveals that both monoclonal and polyclonal antibodies cross-react to a protein with an apparent molecular mass of 50 kDa in an E. harpa cell extract and to a protein of 49 kDa in a macronuclear extract of E. eurystomus. The antibodies used in this study have no effect upon in vitro DNA synthesis in the replication band of E. eurystomus.

Detection of antigenic cyst wall elements in Colpoda inflata: an immunoelectron microscopic study and immunoblotting identification of cyst wall polypeptides.

By using an antiserum against isolated cyst walls from resting cysts of the ciliate Colpoda inflata, cyst wall polypeptides have been identified by immunoblotting test. Likewise, an immunoelectron microscopical study on both complete resting cysts and isolated cyst walls to localize the cyst wall proteins recognized by the antiserum, has been carried out. The immunoblotting test showed that three main polypeptide bands were recognized by the antiserum, with tentative molecular weights of 61, 66 and 70 kDa respectively. This methodology provides a better identification of cyst wall proteins after electrophoretic separation of cyst wall samples from ciliate resting cysts.

Purification, characterization, and amino acid sequence of the mating pheromone Er-10 of the ciliate Euplotes raikovi.

The mating pheromone Er-10 from mat-10 homozygous Euplotes raikovi was purified by a three-step purification procedure with an overall yield of 62%. It was identified as a protein of molecular weight 8000 having an isoelectic point of 3.9. Its complete primary structure was determined by automated Edman degradation of the whole protein after performic acid oxidation and of peptides generated by cyanogen bromide and Staphylococcus aureus V8 protease. The proposed sequence is Asp1-Leu-Cys-Glu-Gln-Ser-Ala-Leu-Gln-Cys10-Asn-Glu-Gln-Gly-Cys-His -Asn-Phe-Cys- Ser20-Pro-Glu-Asp-Lys-Pro-Gly-Cys-Leu-Gly-Met30-Val-Trp-Asn- Pro-Glu-Leu-Cys- Pro38. The calculated molecular weight of 4191.7, which is in good agreement with the value of m/z 4190.7 obtained by fission fragment ionization mass spectrometry, suggests that the native structure is a dimer with three intrachain disulfide bonds in each subunit. The amino acid sequence is 43% identical with that of the E. raikovi mating pheromone Er-1, with the identities concentrated in the amino-terminal half. The half-cystine locations are conserved, but Er-10 is two residues shorter than Er-1. Prediction of the secondary structure suggests that Er-10 may also contain a helical structure at the amino terminus. These results indicate that the mating pheromones of E. raikovi form a homologous family.

Differential distribution of alpha-tubulin isotypes in Euplotes eurystomus determined by confocal immunofluorescence microscopy.

Detergent permeabilized Euplotes eurystomus (a fresh water hypotrichous ciliate) was reacted with monoclonal and polyclonal antibodies specific for either detyrosinated or tyrosinated alpha-tubulin (Glu- or Tyr-tubulin). The isolated cytoskeleton-nuclear complex was examined by Western immunoblotting and by immunofluorescent and electron microscopic methods. Both Glu- and Tyr-tubulins were detected by immunoblot analysis. Immunofluorescent microscopy indicated that the alpha-tubulin isotypes are concentrated in different regions of permeabilized cells: Glu-tubulin is located primarily in cirri, membranelles, and surrounding the macro- and micronuclei. Tyr-tubulin is principally at the bases of cirri and membranelles. This differential distribution of alpha-tubulin isotypes is discussed in terms of current concepts concerning the correlation of tubulin post-translational modifications to microtubule stability. Confocal immunofluorescent imaging was of critical importance in clearly differentiating the Glu-tubulin isotype surrounding the macro- and micronuclei from a brilliantly fluorescent environment originating from cytoskeletal structures. In conjunction with conventional and stereo-electron microscopy, confocal optical microscopy provided convincing evidence for a "basket" of microtubules surrounding both nuclei.

Lectin-like components in the macronuclear replication bands of Euplotes eurystomus.

Upon incubation with fluoresceinylated neoglycoproteins, isolated macronuclei from the ciliated protozoan Euplotes eurystomus display different labelling patterns depending on the nature of the sugar bound to the neoglycoproteins. Specific sugar-binding components (i.e., lectin-like molecules) are associated with presumed nucleoli and with the macronuclear replication bands. This is the first demonstration that DNA synthesis and sugar-binding components are co-localized in an eukaryotic cell.

Replication bands and nucleoli in the macronucleus of Euplotes eurystomus: an ultrastructural and cytochemical study.

The principal structural compartments of the macronucleus of Euplotes eurystomus were examined by ultrastructural and cytochemical procedures. Interphase chromatin is condensed in highly compact granules that stain intensely with the DNA-specific osmium-amine procedure. Nucleoli react strongly with silver and with thiol-specific reagents, but are almost completely unstained by osmium-amine. The organelle of DNA synthesis, the replication band, is composed of 2 zones. The forward zone consists of highly ordered chromatin fibers, stains strongly with osmium-amine, with silver, and with thiol-specific reagents. The rear zone, which is the site of DNA synthesis, is impoverished in DNA, and is very sensitive to collapse induced by in vivo heat shock, or during nuclear isolation.

Purification and characterization of new mating pheromones of the ciliate Euplotes raikovi.

Cell union in mating pairs in the ciliate Euplotes raikovi is controlled by a system of multiple mating types which are inherited with alleles codominant at the genetic locus mat and expressed via diffusible mating pheromones. The mating pheromones Er-2, Er-3, and Er-11 were purified from cells homozygous for the mat-2, mat-3, and mat-11 alleles, respectively. These pheromones are proteins of similar Mr (11,000-12,000) and acidity (pI 3.7-4.0) and are active at a concentration that varies from 2.9 X 10(-12) to 1.2 X 10(-11) M. Data on amino acid composition revealed that an unusually high amount of cysteine (12-15.7%) and poor contents of basic amino acids are common to every pheromone. On the basis of this uniformity in the main biochemical traits, which also holds for the previously purified pheromone Er-1, it was concluded that E. raikovi mating pheromones are members of a family of proteins structurally diversified from each other to varying extents.

Isolation of the mating-inducing factor of the ciliate Euplotes.

Numerous strains of different mating types of the marine ciliate Euplotes raikovi have been found to be autonomous excreters into the surrounding medium of specific mating-inducing factors (gamones) (Luporini, P et al., J exp zool 226 (1983) 1 [9]). The gamone from the mating type represented by strain 13 has been isolated and identified as a glycoprotein with a molecular weight (MW) of about 12 kD and a pI of 4. It has been termed euplomone r 13. At a concentration of 3 X 10(-12) M, euplomone r 13 specifically induces cells of a complementary mating type to unite in conjugation within 2 h.

2-Aminoethylphosphonic acid concentrations in some rumen ciliate protozoa.

The concentration of 2-aminoethylphosphonic acid has been measured in seven genera of rumen ciliate protozoa. Expressed as milligrams per gram of total nitrogen, 2-aminoethylphosphonic acid concentrations ranged from 17.2 in Ophryoscolex spp. to 72.4 in Eremoplastron spp.

The size and structure of the DNA genome of symbiont xenosome particles in the ciliate Parauronema acutum.

The size and structure of the DNA genome of xenosomes, bacterial endosymbionts of the marine hymenostome ciliate, Parauronema acutum 110-3, were investigated. Renaturation kinetic measurements, determined optically and by hydroxyapatite chromatography, suggested a genome size of 0.34 x 10(9) daltons. Sedimentation rate measurements of DNA gently released from the symbionts yielded molecules of comparable size. The analytical complexity, determined chemically, was 3.03 x 10(9) daltons. Consistent with these and other data is a model for the structure of the symbiont genome in which the DNA exists in the form of nine circularly permuted, double-stranded DNA molecules of unique sequence, each of molecular weight 0.34 x 10(9). It is suggested that xenosomes and certain symbionts found in ciliated protozoa may be extant forms of once free-living bacteria that have adapted to the intracellular environment.

Evidence for the lack of actin involvement in mitosis and in the contractile process in Spirostomum teres.

Studies using actin decoration techniques and electron microscopy have failed to show the presence of actin in the ciliate Spirostomum teres. The internal and external membranes of dividing cells were made permeable by treatment with Triton. The myosin subfragment S-1 was introduced into the cells for incubation under conditions suitable for actin decoration. Arrowhead decoration of microfilaments was not observed within dividing micronuclei or in cytoplasmic filament bundles. However, using similar procedures, the brush border of mouse small intestine yielded clearly decorated microfilaments. A spectrophotometric DNase I inhibition assay specific for G-actin demonstrated that the level of actin in S. teres extracts was less than 0.06% of the total soluble protein and confirms the observations made using the decoration procedures. On the basis of this work, it appears that actin does not play a significant part in either mitotic chromosome movement or contractility in S. teres.

Antibodies against Z DNA react with the macronucleus but not the micronucleus of the hypotrichous ciliate stylonychia mytilus.

Using indirect immunofluorescence, we studied the reaction of antibodies specific for left-handed Z DNA with the nuclei of the hypotrichous ciliate Stylonychia mytilus. In the vegetative cell, the macronucleus reacts strongly with these antibodies, but no reaction can be detected with micronuclei. However, an antibody that binds to denatured and right-handed B DNA reacts with both types of nuclei. No reaction of the anti-Z DNA antibody is seen in the macronuclear replication band. Digestion of macronuclei with DNAase I leads to a decrease in the anti-Z DNA antibody reaction. Some stages of the developing macronucleus were also investigated. No reaction is seen at the polytene chromosome stage, but following DNA elimination the nucleus is seen to react with the antibody.

Extraction and some properties of the proteins, Spastin B, from the spasmoneme of Carchesium polypinum.

Proteins of the contractile spasmoneme from Carchesium polypinum were extracted in 2% SDS, 30% acetic acid, or 8 M urea. The proteins extracted in SDS had a wide molecular weight distribution when examined by SDS-polyacrylamide gel electrophoresis. On the other hand, the proteins extracted in urea and acetic acid had three major peaks with molecular weights of about 16,000, 18,000, and 22,000. Most of these proteins were soluble even in the absence of urea and furthermore were found to be monomeric, since the sedimentation coefficient, s20,w, measured by analytical ultracentrifugation was 2.0S. The electrophoretic mobility of the proteins extracted in urea or in acetic acid was examined on alkaline gels. In the presence of free Ca2+, the mobility was significantly reduced compared with that in the absence of free Ca2+. These Ca-binding proteins were heat-stable and could not interact with troponin I. The implications of these proteins and others in relation to the contractility of the spasmoneme in Carchesium stalk are discussed.

Higher order DNA structure in macronuclear chromatin of the hypotrichous ciliate Oxytricha nova.

On lysis of macronuclei from the ciliated protozoan Oxytricha at 0.5-2 M NaCl, the DNA, which is normally found as discrete molecules ranging from 0.5 to 20 kilobases, appears in high molecular weight aggregates. Various treatments of the macronuclear lysate (i.e., nucleases, proteases, variation of salt, pH, and temperature) indicate that preservation of the aggregate structure depends on both nucleic acid-nucleic acid and nucleic acid-protein interactions. Purification of the DNA-protein complex after lysing the nuclei in 2 M NaCl shows that one major nuclear protein copurifies with the DNA. As shown by DNA-protein binding experiments, this protein has a high affinity for DNA; however, no evidence for sequence specificity of the protein binding was obtained. Chromatin reconstitution experiments suggest that the protein in itself is not sufficient for DNA aggregation in nuclei, but other factors, possibly the native chromatin structure, are required. Electron microscopy of the purified DNA-protein complex showed structures similar to those observed previously with in vitro-aggregated purified macronuclear DNA (14). A model is presented in which the terminal inverted repeat sequences found on all macronuclear DNA molecules interact with each other forming multistranded DNA complexes. The formation of these structures may be accelerated and stabilized by a protein in vivo.

Spectroscopic characterization of the Stentor photoreceptor.

1. On the basis of chromatographic and spectroscopic (absorption, fluorescence and its polarization, fluorescence lifetime, circular dichroism) characterization of the Stentor photoreceptor (stentorin) for photophobic response, the photoreceptor chromophore released from mild acid hydrolysis has been identified as hypericin. 2. The native chromophore is apparently linked to a protein (65 K) containing Lys and several hydrophobic residues, which is soluble in acetone and n-pentane. The peptide-linked stentorin (I) chromophore exhibits circular dichroism in the visible region due to the induced optical activity provided by the peptide. 3. The sodium dodecyl sulfate polyacrylamide gel electrophoresis of a 38% fraction of the sucrose density centrifugation has resolved stentorin II proteins having molecular weights of 13 000, 16 000, 65 000 and 130 000. These proteins, as well as the acetone-soluble peptide, have been spectroscopically characterized with particular emphasis on their primary photoreactivity as the photophobic receptor of Stentor coeruleus. 4. Irradiation of whole living Stentor in dilute buffer solutions induces a decrease in the pH of the medium. A strong dependence upon pH in the fluorescence spectra of both synthetic and native chromophores is also evident, showing a significant drop in the pKa of one or more hydroxyl groups in the excited state. A mechanism for the photophobic response, based on this lowering of the pKa as the primary photoprocess, has been discussed.

[Characteristics of DNA in vegetative specimens of Bursaria truncatella]. / Kharakteristika DNK vegetativnykh osobei infuzorii Bursaria truncatella.

DNA from Bursaria truncatella was isolated and purified by conventional methods. The DNA base content was calculated from both centrifugation in CsCl and melting. The GC-content is 24%. In CsCl density gradient 3H-DNA is banded as a single peak at a range 1.682--1.688 g/cm3 with the maximum at 1.684 g/cm3. The Tm in 0.12 M FB (pH 6.8) was 79 degrees C. About 50% of DNA seems to be represented by highly repetitive sequences, another 50% being made of single-copy sequences (a preliminary data on DNA-DNA reassotiation kinetics). For the estimation of the molecular weight of DNA, the cells were lysed immediately before the centrifugation, at the surface of the alkaline isokinetic sucrose gradient solution. Two components of DNA were detected with molecular weights 10-10(6) and 100-10(6) daltons. It is likely that these two components belong to the macronuclear DNA, because according to cytophotometrical evidence the DNA content in the macronucleus of B. truncatella is 2500 times as much as that in its micronucleus.