The Membrane Method of Electron Microscope Autoradiography

Thin sections of methacrylate and Araldite embedded tissues labelled with radioactive isotopes were transferred with a wire loop or brush from the knife edge onto thin formvar membranes which covered 7 mm holes in 76 × 25 × 1.5 mm or 76 × 38 × 1.5 mm plastic slides. To facilitate the mounting of sections, a platform supported the plastic slides close to the ultramicrotome knife. Photographic emulsion diluted 1:5 or 1:10 with water was applied with a pipette to the upper surface of each formvar membrane to cover the mounted sections. Excess emulsion was drained off and the remaining thin film was dried on a warm plate at 45 C to produce a uniform layer over the sections. After storing in the dark for several weeks, preparations were processed in photographic solutions and washed, and sometimes stained, before applying electron microscope grids to the underside of each formvar membrane. To detach each grid with its adherent formvar, section and emulsion, the membrane was pierced around the perimeter of the grid. Grain counts made over nuclei of cells labelled with tritiated thymidine indicate that emulsion is uniformly distributed over each section and that quantitative comparison is possible between labelled areas.     

Intracellular Localization of Nigeran in the Wall of Aspergillus aculeatus by Autoradiography with the Electron Microscope

Aspergillus aculeatus mycelium was incubated with [(3)H]glucose under conditions that promote nigeran (mycodextran) accumulation (low pH, in the absence of nitrogen). Autoradiography revealed that essentially all the (3)H was localized around the hyphal perimeter. The results strongly support a hyphal wall location for nigeran.     

Evolution of the Germline Actin Gene in Hypotrichous Ciliates: Multiple Nonscrambled IESs at Extremely Conserved Locations in Two Urostylids

In hypotrichous ciliates, macronuclear chromosomes are gene‐sized, and micronuclear genes contain short, noncoding internal eliminated segments (IESs) as well as macronuclear‐destined segments (MDSs). In the present study, we characterized the complete macronuclear gene and two to three types of micronuclear actin genes of two urostylid species, i.e. Pseudokeronopsis rubra and Uroleptopsis citrina. Our results show that (1) the gain/loss of IES happens frequently in the subclass Hypotrichia (formerly Stichotrichia), and high fragmentation of germline genes does not imply for gene scrambling; and (2) the micronuclear actin gene is scrambled in the order Sporadotrichida but nonscrambled in the orders Urostylida and Stichotrichida, indicating the independent evolution of MIC‐actin gene patterns in different orders of hypotrichs; (3) locations of MDS–IES junctions of micronuclear actin gene in coding regions are conserved among closely related species.     

Effects of Glutaraldehyde and Osmium Tetroxide on Hypotrichous Ciliates, and Determination of the Most Satisfactory Fixation Methods for Electron Microscopy

SYNOPSIS. In electron microscope studies on hyppotrichous ciliates, cytolysis and/or body deformation–resulting from insufficient contact with glutaraldehyde and poor infiltration of Epon 812, particularly into the buccal cavity, usually were observed. Fixation experiments were carried out to examine the effects of some fixatives on Euplotes eurystomus, Oxytricha bifaria and Stylonychia mytilus to establish the best fixation technic applicable to all species of hypotrichous ciliates. Although the effects of fixation varied considerably with the species, 2 fully satisfactory fixation methods were developed by using OsO₄ and glutaraldehyde. In one, a mixture of both fixatives was employed; in the other a very short application of OsO₄ was followed by glutaraldehyde. The problem of infiltration was solved by using Spurr's low-viscosity embedding medium in place of Epon 812.     

Phylogenetic Relationships Among Hypotrichous Ciliates Determined with the Macronuclear Gene Encoding the Large, Catalytic Subunit of DNA Polymerase α

The complete macronuclear DNA polymerase α gene, previously sequenced in Oxytricha nova, has been cloned from a genomic macronuclear library and sequenced for the hypotrich O. trifallax. Macronuclear DNA clones of DNA polymerase α encoding ∼1000 amino acids, or approximately two-thirds of the open reading frame, have been obtained by PCR and sequenced for Halteria grandinella, Holosticha species, Paraurostyla viridis, Pleurotricha lanceolata, Stylonychia lemnae Teller, Sty. mytilus, Uroleptus gallina, and Urostyla grandis. Phylogenetic relationships inferred from DNA polymerase α amino acid sequences have been used to clarify taxonomic relationships previously determined by morphology of the cell cortex. Hypotrich phylogenies based on DNA polymerase α amino acid sequences are incongruent with morphological and other molecular phylogenies. Based upon these data, we assert that, contrary to morphological data, O. nova and O. trifallax are different species, and we propose that the oligotrich Halteria grandinella be reclassified as a hypotrich. This work also extends the available data base of eukaryotic DNA polymerase α sequences, and suggests new amino acid sequence targets for mutagenesis experiments to continue the functional dissection of DNA pol α biochemistry at the molecular level. Received: 7 January 1997 / Accepted: 7 April 1997     

一种用于观察纤毛虫表膜下结构的扫描电镜样品制备新方法

该实验摸索出通过扫描电镜观察纤毛虫表膜下三维结构的新方法:用适当浓度的KMnO_4作为固定剂,固定虫体细胞表膜,调整固定液的渗透压使细胞在低渗溶液中胀破、细胞质溶出,表膜剥落下来、内外翻转,经脱水、冷冻干燥、喷金后,在扫描电镜下对爽口虫(Climacostomumsp.)、尾草履虫(Paramecium caudatum)及拟尾柱虫(Paraurostyla weissei)的表膜下结构进行了观察。结果表明:利用此方法能够观察到表膜下层次分明而又清晰的三维立体构象。此方法可为纤毛虫表膜及其它细胞质膜的研究提供可借鉴的样品制备新方法。     

Evidence of Extensive Fragmentation of Macronuclear DNA in Two Non-Hypotrichous Ciliates

ABSTRACT. Total cellular DNA from the ciliates Halteria grandinella and Trithigmostoma cucullulus was analyzed by agarose gel electrophoresis. The macronuclear DNA (MAC DNA) of Halteria consisted of very small fragments, which suggests that the MAC DNA organization of oligotrichs resembles that of hypotrichs (gene-sized DNA). The MAC DNA of Trithigmostoma , a cyrtophorid having a heteromeric MAC, also existed as small fragments, but with a significant fraction (20–30%) comprising larger molecules unresolved by the method used. It is suggested that MAC heteromery is related to the differential localization of two kinds of DNA molecules of different sizes.     

Morphological Features of DNA Macromolecules as Seen with the Electron Microscope

Desoxyribosenucleic acid molecules isolated from salmon sperm were studied with the electron microscope. The essential step in the technique which makes it possible to visualize the individual molecules consists in a preparative step wherein the materials are supported on the extremely smooth surface of cleaved mica where they are shadow-cast with platinum, which is then backed with a supporting film and stripped for observation in the usual manner. The DNA, which was originally about 8 million molecular weight, was also examined after fragmentation by sonic vibration. The fragments show a certain degree of rigidity and the ends generally terminate abruptly, indicating that the double helices of the Watson-Crick model both break close to the same place. DNA molecules heated to temperatures between 90 and 100°C, coil up into amorphous patches, although a few apparently unaltered molecules survive such heating.     

Inhibition of Dna Synthesis In the Macronuclear Replication Band of Euplotes Eurystomus

ABSTRACT. The replication band is a large, migrating, macronuclear domain that is the site of DNA synthesis in hypotrichous ciliated protozoa. A number of agents that produce inactivation of this structure and its replicational activity are described here. These agents include heat shock, aphidicolin, cell crowding, various cAMP phosphodiesterase inhibitors and a calmodulin inhibitor. With the exception of aphidicolin, which has a direct inhibitory effect upon DNA polymerases, the mechanisms of inactivation are presently unknown. the inactivating properties of cAMP phosphodiesterase inhibitors suggest that intracellular cAMP levels may influence replication band structure and function.     

Morphogenetic Pattern in Laurentiella acuminata (Ciliophora,Hypotrichida): Its Significance for the Comprehension of Ontogeny and Phylogeny of Hypotrichous Ciliates1

Morphogenetic events during division, physiological reorganization, and postraumatical regeneration, the last being induced both chemically and microsurgically, were studied by light microscopy on protargol-impregnated specimens of the hypotrichous ciliate, Laurentiella acuminata. Parakinetal stomatogenesis, from transverse cirrus-1 during division and reorganization, changes during regeneration to a parakinetal one which characterizes more primitive members of Hypotrichida in the S. O. Stichotrichina, but solely when the AZM is damaged. These morphogenetic events a) confirm the previous inclusion of L. acuminata among the Oxytrichidae on the basis of its morphological characters and indicate that it is a primitive species of this family related with the Stichotrichina through genera Pleurotricha and Paraurostyla; b) suggest a synthetic model that explains both the positioning and timing of cortical morphogenesis in the cell cycle. The key point of this model is the attribution to the AZM of a repressive capacity on the stomatogenic area, the last one being positioned according to the system of gradients of morphogenetic activity proposed by Jerka-Dziadosz to explain location of primordia in urostylids. This repression is manifested not as a gradient, as indicated by De Terra, but as a long-term repression limited to a certain distance. Simultaneous repression and stimulation occurring in a growing cortex with the AZM remaining constant in size could explain the critical ratio, buccal cortex/somatic cortex, at which stomatogenesis is triggered as indicated by De Terra.     

Looking at DNA through the Electron Microscope: the Work of Jack Griffith

Electron Microscopic Visualization of the RecA Protein-mediated Pairing and Branch Migration Phases of DNA Strand Exchange(Register, J. C., 3rd, Christiansen, G., and Griffith, J. (1987) J. Biol. Chem. 262, 12812–12820)Formation of DNA Loop Replication Fork Generated by Bacteriophage T7 Replication Proteins(Park, K., Debyser, Z., Tabor, S., Richardson, C. C., and Griffith, J. (1998) J. Biol. Chem. 273, 5260–5270)Jack D. Griffith was born in Logan, Utah, in 1942. He attended Occidental College in Los Angeles and received his B.A. in physics in 1964. Griffith then enrolled at the California Institute of Technology where he worked with Journal of Biological Chemistry (JBC) Classic author James Bonner (1) studying chromosome structure with electron microscopy (EM).Open in a separate windowJack GriffithIn the late 1960s, scientists were using a technique called metal shadow casting to visualize molecules via EM. The method involved spraying a layer of metal on the molecule. Because the sample was slightly raised, it got coated with more metal than its supporting film, which allowed the molecule''s outline to be seen with an electron microscope. A variation on the technique in which the sample was coated with denatured protein was used to visualize DNA. This provided a good way to look at the shape of DNA, but the specific proteins bound to the DNA were obscured by the thick coating. For his Ph.D. work, Griffith developed the EM technology needed to directly visualize bare DNA and DNA-protein complexes. His methods involved carefully controlled rotary shadow casting with tungsten and mounting the DNA on very thin carbon films.After graduating in 1969, Griffith did a 1-year postdoctoral fellowship with Benjamin Siegel at Cornell University and a 3-year fellowship with JBC Classic author Arthur Kornberg (2) at Stanford University. Using the methods Griffith developed at Caltech, Griffith, Kornberg, and Joel A. Huberman published a paper showing an EM image of Escherichia coli DNA polymerase I bound to DNA (3). This was not only the first EM image of DNA bound to a known protein, but it also showed that electron microscopy had the potential to provide quantitative information about macromolecular assemblies involving DNA.Griffith stayed at Stanford as a research scientist until 1978 when he became an associate professor at the Lineberger Comprehensive Cancer Center and the Department of Microbiology and Immunology at the University of North Carolina at Chapel Hill. At UNC, he designed a research program in which he used EM and biochemical tools to study DNA. The two JBC Classics reprinted here demonstrate some of those studies.In the first Classic, Griffith and his colleagues investigated the role of the E. coli RecA protein in homologous recombination. RecA catalyzes this process by promoting pairing and strand exchange between homologous DNA molecules. The scientists used EM to follow reactions in three homologous DNA pairs: supertwisted double-stranded (ds) DNA and linear single-stranded (ss) DNA; linear dsDNA and circular ssDNA; and linear dsDNA and colinear ssDNA. They found that all three reactions undergo a three-step pathway. First, the RecA protein-ssDNA filament makes contact with a homologous dsDNA (joining). Second, both DNA partners are at least partially enveloped within the nucleoprotein filament, and if the DNA topology is favorable, exchange of DNA strands then ensues (envelopment/exchange). Finally, upon completion of strand exchange, this complex is resolved and the products are released.In the second Classic, Griffith teamed with JBC Classic author Charles C. Richardson (4) and used EM to examine the architecture of the DNA and DNA-protein intermediates involved in replication reactions employing the T7 replication proteins. This study showed the first direct evidence of the presence of a DNA loop at the replication fork and provided a long sought after proof of the Alberts trombone model of looping of the lagging strand during replication. One of Griffith and Richardson''s co-authors on this paper, Stanley Tabor, is the son of long time JBC editor Herbert Tabor.Griffith remains at the University of North Carolina at Chapel Hill as Kenan Distinguished Professor of Microbiology and Immunology and Biochemistry. He has received many honors and awards for his contributions to science including the Ellison Senior Scholar Award (2001–2005), the ASBMB Herbert A. Sober Award (2002), the Grand Gold Medal of Comenius University, Slovak Republic (2006), and the Glenn Foundation Award (2007). He was also elected to the American Association for the Advancement of Science (2001) and the American Academy of Arts and Sciences (2005) and served on the Journal of Biological Chemistry editorial board from 2002 to 2007.     

Electron Microscopy of Adenovirus 12 Replication II. High-Resolution Autoradiography of Infected KB Cells Labeled with Tritiated Thymidine

Incorporation of tritiated thymidine by KB cells infected with oncogenic adenovirus 12 was studied by means of high-resolution electron microscopic autoradiography. After a 1-hr pulse with tritiated thymidine, infected and control cultures were fixed at 8, 16, 24, 30, and 36 hr. Infected cultures showed a higher percentage of labeled cells. During early stages, the frequency of silver grains in the nucleus and in the nucleolus was higher in infected material. From 24 hr on, there was an inhibition of nuclear and nucleolar deoxyribonucleic acid (DNA) synthesis. At late stages, one-third of the label was located over nuclear inclusions, type II and IV, previously shown to be composed of DNA and protein, while a large majority of the remaining grains were located over the nucleoplasm. The possibility is considered, that the early increase in nuclear and nucleolar DNA synthesis produced by adeno 12 replication could in part be due to newly synthesized cellular DNA, as has been reported by others with respect to other oncogenic DNA viruses.     

Replication of Bacteriophage ST-1 in Escherichia coli Strains Temperature Sensitive in DNA Synthesis

Bacteriophage ST-1 replication requires DNA polymerase III (dnaE) but not DNA polymerases I or II, DNA ligase, or the products of dnaA, B, or C-D. It was not certain whether dnaG was required. These results differ considerably from those reported for X-174.     

Replication process of the parvovirus H-1. VII. Electron microscopy of replicative-form DNA synthesis.

The geometry of replicative form (RF) DNA synthesis of the H-1 parvovirus was studied with the electron microscope using formamide or aqueous variations of the Kleinschmidt spreading procedure. H-1 DNA was isolated from human or hamster cells infected with a temperature-sensitive mutant, ts1, which is deficient in progeny single-stranded DNA synthesis at the restrictive temperature (S.L. Rhode, 1976), thus minimizing possible confusion between RF and progeny DNA replicative intermediates (RIs). The purity of the isolated H-1 DNA, as determined by gel electrophoresis, ethidium bromide staining, autoadiography, and digestion with endo R-EcoRI, was high. H-1 RF DNA'S WERE LINEAR DOUBLE-STRANDED MOLECULES, 1.53 MUM IN LENGTH. H-1 RIs of RF DNA replication were double-stranded, Y-shaped molecules, with the same length as RF DNAs. The replication origin was localized no more than 0.15 genome lengths from one end of the RF DNA, with replication proceeding toward the other end at a uniform rate. Similar RF and RI molecules of dimer size were also observed. The length of H-1 single-stranded DNA extracted from purified virions was measured relative to that of phiX174 and it had a very similar contour length, so that the molecular weight of H-1 single-stranded DNA would be at least 1.48 X 10(6) to 1.59 X 10(6) (Berkowitz and Day, 1974).     

Replication Process of the Parvovirus H-1 III. Factors Affecting H-1 RF DNA Synthesis

Replication of the single-stranded DNA parvovirus H-1 involves the synthesis of a double-stranded DNA replicative form (RF). In this study, the metabolism of RF DNA was examined in parasynchronous hamster embryo cells. The initiation of RF DNA replication was found to occur late in S phase, as was the synthesis of the DNA upon which subsequent viral hemagglutinin synthesis is dependent. Evidence is presented which indicates that initiation of RF replication requires proteins synthesized in late S phase, but that concomittant protein synthesis is not required for the continuation of RF replication. The data also suggest a requirement for viral protein(s) for progeny strand synthesis. Incorporation of 5-bromo-2'-deoxyuridine (BUdR) into viral DNA resulted in an "all-or-none" inhibition of viral hemagglutinin and viral antigen synthesis. BUdR inactivation of viral protein function was used to explore the time of synthesis of viral DNA serving as template for viral RNA synthesis and the effect of viral protein on RF replication and progeny strand synthesis. Results of this study suggest that parental RF DNA is synthesized shortly after infection, and that viral mRNA is transcribed from only a few copies of the viral genome in each cell. They also support the conclusion that viral protein is inhibitory to RF DNA replication. Density labeling of RF DNA with BUdR, allowing separation of viral strand DNA (V) from viral complementary strand (C), provided additional data in support of the above findings.     

Zoosporulation in Labyrinthula sp.; an Electron Microscope Study1

SYNOPSIS. Zoosporulation in Labyrinthula sp. in monoxenic culture was initiated by aggregation of spindle cells into reticulate sori. The spindle cells then changed into rounded or oval cells and formed, de novo, 2 pairs of centrioles at opposite sides of each nucleus. A pair of granular aggregates (protocentrioles) ～ 240 mμ in diameter served as precursor bodies during centriole formation. Spindle microtubules around the prophase nucleus connected the pairs of centrioles but were not found in the nucleoplasm until nuclear envelope fragmentation occurred. Prophase nuclei of uninucleated sporangia contained synaptinemal complexes; therefore, meiosis is presumed to occur. The envelope fragments moved toward the centrioles and regrouped to form the nuclear membranes of the daughter cells. Alternating nuclear and cytoplasmic divisions subdivided the preparation into 8 cells which differentiated into laterally biflagellated zoospores. Flagellar development involved growth of the kinetosome microtubules into a bud which formed over the kinetosome tangential to the cell surface. Kinetosomes were derived directly from centrioles with little differentiation other than addition of an electron-dense core to the lumen of the centriole. Zoospore ultrastructure included a stigma comprised of a row of electron-dense granules located slightly under the plasmalemma and posterior to the pair of kinetosomes. A single row of 17–21 microtubules lay parallel to the stigma granules, one or more being connected to the anterior kinetosome. A striated fiber apparatus similar to that found in some phytoflagellates connected the midregions of the kinetosomes. Fibers 1.0–1.2 μ long were attached to the plasmalemma around the base of the anterior flagellum. Zoospores settled on the substrate and differentiated directly into spindle cells. Since synaptinemal complexes were observed the planonts are probably haploid zoospores and probably not gametes since planogametic copulation was not observed.