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.     

Examination of the macronuclear replication band in Euplotes eurystomus with monoclonal antibodies

A panel of eight monoclonal antibodies (MAbs) was prepared from spleen cells of mice immunized with macronuclear replication bands (RBs) isolated from Euplotes eurystomus. Antibodies were investigated with a solid phase radioimmunoassay (RIA) using either soluble chromatin from isolated RBs or from total macronuclei as antigen. The RIA showed that several MAbs recognized antigens present only in the RB or macronucleus, whereas others recognized antigens present in both structures. Specificity of the MAbs was also examined by indirect immunofluorescence. Antibody C10 recognized an antigen in the rear zone of the RB, whereas MAbs G6 and B2 appeared to stain both the forward and rear zones of the RB. Antibody A7 recognized an epitope distributed throughout the macronucleus except in the RB. Cytochemical studies with degradative enzymes suggested that antigens localized by immunofluorescence were composed of proteins. Immunoblots of SDS PAGE permitted identification of a few proteins that reacted with three of the RB-specific MAbs. Monoclonal antibodies that identify the presence or absence of reactivity of specific proteins in the RB could prove useful in the study of chromatin structure and the mechanism of chromatin replication.     

In vitro DNA synthesis in the macronuclear replication band of Euplotes eurystomus

Isolated macronuclei from the hypotrichous ciliated protozoan Euplotes eurystomus incorporate biotinylated dUTP specifically into the replication band (RB) as detected with immunofluorescence, using rabbit anti-biotin antibodies followed by fluorescein-conjugated goat anti-rabbit IgG. When gold-conjugated goat anti-rabbit IgG was used in a preembedded reaction, subsequent immunoelectron microscopic analysis demonstrated that the biotinylated nucleotide appeared more concentrated in the rear zone of the RB, with almost no labeling in the forward zone. It was possible to use the immunofluorescent assay to establish that incorporation of biotinylated dUTP is inhibited by simultaneous addition of N-ethyl maleimide or aphidicolin, and by omission of any one of the other unlabeled dNTPs. In addition, prolonged heat shock of the intact cells, before lysis and in vitro assay, yielded markedly reduced incorporation. Comparison with published data on the in vivo incorporation of [3H]thymidine into Euplotes eurystomus RBs indicates the fidelity of the in vitro reaction.     

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.     

Ultrastructure of Pellicular and Ciliary Structures of Euplotes eurystomus

SYNOPSIS. Many of the sub-pellicular and infraciliary structures in protozoa have proved difficult to study with standard thin-sectioning technics. When these structures are viewed in isolated and fragmented form, many of the thin-sectioning difficulties are circumvented. Langmuir-trough isolation followed by critical-point drying, as well as thin sectioning, were used in this study to determine the patterns of sub-pellicular microtubules and fibrils interconnecting kinetosomes of membranelles and cirri of Euplotes eurystomus. The fibrillar network in the bases of these ciliary organelles is presented in some detail and apparent variations in pattern are noted. Functional aspects of some of the structures are discussed. With special preparation nearly whole Euplotes may be obtained for study in the electron microscope. Fused cilia were frequently obtained and their ultrastructure was studied.     

An H1-like protein from the macronucleus of Euplotes eurystomus

An H1-like protein has been purified from the macronucleus (MAC) of the hypotrichous ciliated protozoan, Euplotes eurystomus. It is present in amounts comparable to the inner histones and is extracted by treatment with 5% perchloric acid or 0.65 M NaCl, but not by 0.35 M NaCl. Treatment of soluble MAC chromatin with the ionic exchange resin AG 50W-X2 in 80 mM NaCl removes MAC H1 and yields H1-depleted chromatin. Mac H1 is lysine-rich and deficient in acidic amino acids. The stoichiometry of the H1 protein is reduced in mononucleosome preparations, consistent with its postulated interaction with linker DNA regions. Thermal denaturation and circular dichroism studies reveal that H1-depleted chromatin contains a larger portion of destabilized DNA than control chromatin. The molecular weight of Euplotes MAC H1 is significantly smaller than most reported H1 proteins. Comparisons are made with extracts of macronuclei from other hypotrichous ciliated protozoa and published reports of other lower eukaryotes.     

Isolation and characterization of chromatin from Caulobacter crescentus

The subunit structure of Caulobacter crescentus chromatin has been proven by electron microscope studies. The use of EDTA-Na2 during the purification of the chromatin complex enhanced the removal of contaminating ribosomes and non-chromatin proteins. The preparation obtained by modified procedure contained RNA polymerase as one of the major proteins and three histone-like proteins (10 K, 17 K and a hitherto not described protein with mol. wt 14 K).     

Isolation and characterization of chromatin subfractions from rat liver

Rat-liver chromatin was digested with micrococcal nuclease at low ionic strength in the presence of a low concentration of CaCl2. The nuclease digest was successfully separated into three fractions, P1, P2, and P3, by gel filtration on a column of Sepharose 2B. P1 fraction was shown to be a mixture of long fragments of partially digested chromatin by the sedimentation profile or by electrophoresis of DNA. P2 fraction contained four histones H2A, H2B, H3, and H4 in almost equal amounts, together with nonhistone protein of low molecular weight. The DNA was composed of three or four fragments less than 300 base pairs long. From the Kav value of the P2 fraction, the average size was estimated to be about 240 base pairs. On analytical ultracentrifugation, this fraction exhibited a monophasic boundary and a sedimentation value of 13.7S. P3 fraction contained nonhistone proteins which showed a molecular weight larger than that of H1 histone. The size of DNA was estimated to be less than 50 base pairs from the Kav value. Based on these results, the P2 fraction was concluded to consist of nucleosome monomer enriched in nonhistone proteins. The P3 fraction is presumably the nuclease-sensitive or internucleosome portion, which contains small amounts of nonhistone proteins.     

Proliferating cell nuclear antigen/cyclin in the ciliate Euplotes eurystomus: localization in the replication band and in micronuclei

Human autoimmune sera specific for proliferating cell nuclear antigen (PCNA)/cyclin (auxiliary protein for DNA polymerase delta) demonstrated the presence of epitopes within the macro- and micronuclei of the hypotrichous ciliated protozoa Euplotes eurystomus. Tightly bound PCNA/cyclin was localized at the site of DNA synthesis in macronuclei, the rear zone of the replication band. Starvation or heat shock, conditions that reduce macronuclear replication, resulted in a decrease of PCNA/cyclin in replication bands. Micronuclei also exhibited PCNA/cyclin localization which persisted for a large proportion of the vegetative cell cycle and exhibited significant resistance to adverse culture conditions. Immunoprecipitation of 35S-labeled soluble Euplotes proteins with PCNA/cyclin autoimmune sera revealed a spectrum of low molecular mass proteins. PCNA/cyclin-like proteins have now been observed in the widely divergent species: human, rat, amphibian, yeast, and ciliated protozoa.     

The synthesis and distribution of DNA in the macronucleus of Euplotes eurystomus

In an attempt to recognize any ordering of DNA synthesis which might occur in ciliates, the distribution of labelled DNA in the G₁ and S periods of the first, second, and third generations of a synchronized population of Euplotes eurystomus was studied by means of autoradiography. The results presented here show that the replicating DNA which at the time of label incorporation is restricted to a morphologically identifiable narrow region of the nucleus (the replication band), becomes dispersed and is evenly distributed throughout the nucleus. This dispersal of labelled DNA occurs previous to division and is observable throughout the G₁ period of the following generation. During the S phase of this second generation, this previously labelled DNA once again becomes restricted to a small portion of the nucleus. Now, however, it is present at the tips of the macronucleus independently of the position of the replication band. Again the labelled DNA is found to be dispersed during G₁ of the third generation. In the S period of this third generation however the radioactive DNA again appears localized in the replication bands which are found at the same position in the nucleus where they were when the pulse was given two generations earlier. The observations derived from the autoradiographic analysis suggest a non-permanent organization for at least those DNA molecules which are replicated during the first third of the S period. This DNA can be associated in either of two specific locations, replication band or tip, and these two patterns of organization alternate from generation to generation.     

Isolation and partial characterization of gamone 1 of Euplotes octocarinatus

A polypeptide, termed gamone 1, was isolated and purified to homogeneity from culture filtrates of mating type VII of the freshwater ciliate Euplotes octocarinatus. The gamone induces intraclonal conjugation in cells of certain other mating types. The isolation and purification of the gamone was carried out by a combination of two chromatographic steps. The purified gamone was found to be still effective in a concentration of approx. 10⁻¹⁵M.