Hermann Frank

Autolytic enzymes and cell division of Escherichia coli

Abstract The shape of most bacteria is determined by a defined macromolecule which encloses the cell completely. Therefore, in this case, morphogenesis can be studied by analyzing the biosynthesis of this defined type of molecule, a sacculus, which is made up from the polymer murein. The sacculus can be isolated in pure form and can be examined under the electron microscope. We took advantage of this fact to study some topological aspects of murein biosynthesis in Escherichia coli. The growth of the structural determinant of the cell wall, the sacculus, was found to be carried out by several functionally different systems. One is involved in cell elongation, others in cell division; these systems were revealed by their differential sensitivity to penicillin. By use of this antibiotic, we were able to localize the site of action of murein hydrolases which are directly involved in bacterial morphogenesis; the topology and timing of their action are correlated with DNA replication. This procedure revealed zonal growth of the sacculus during cell division. Thus, this structural element of the cell wall is enlarged exactly as predicted by the replicon hypothesis.

Properties of mouse leukemia viruses: III. Electron microscopic appearance as revealed after conventional preparation techniques as well as freeze-drying and freeze-etching

Abstract Rauscher, Friend, and Gross strains of murine leukemia viruses (MuLV) were studied with negative staining, freeze-drying, and freeze-etching techniques. Negative staining of the virus particles with uranyl acetate (UA) and the combination of phosphotungstic acid (PTA) negative staining with freeze-drying showed that the virus particles are roughly spherical and are covered with “knobs” about 80 A in diameter which are weakly bound to the viral membrane. Freeze-etching of the virus particles confirmed the existence of the “knobs.” A diameter of about 1060 A was determined for the virus particles in freeze-dried preparations. Freeze-drying and shadowing of the viral cores liberated by Tween 80 and ether, ether alone or Triton X-100, revealed that their surfaces have a regular pattern of hexagonally arranged subunits with a diameter of about 60 A and a center-to-center distance of about 75 A. The existence of pentons as well as the configuration of the shadows of freeze-dried cores suggested that the viral core possesses icosahedral symmetry. Negative staining of fixed virus particles resolved the core shell into two tracks, a fact which indicates the existence of two components. A smooth membranous layer underlying the layer of regularly arranged subunits was observed in liberated cores. The diameter of the viral cores was about 800 A as determined after freeze-drying. The internal component of the core, the nucleoid, is a filamentous structure, which may possess helical symmetry. Considering the above described substractures, we have proposed a model of MuLV.

Human Teratocarcinomas Cultured in vitro Produce Unique Retrovirus-like Viruses

We have previously reported that among a series of human tumours investigated, only human teratocarcinoma cell lines derived from testicular tumours or pulmonary metastases of patients in Germany and the U.S.A. produced retrovirus-like particles spontaneously, albeit in low amounts. In a recent publication electron microscopical data suggested that the human teratocarcinoma-derived ( HTD ) particles were morphologically closely related, but not identical, to the type C retroviruses of animals. In this communication, the explantation of three human teratocarcinoma cell lines is briefly described. Evidence is presented that HTD particles (i) are synthesized only in a fraction of the epithelioid and differentiating cells; (ii) can be induced biochemically in a manner characteristic of retroviruses; (iii) either are not infectious or possess a peculiar host range; (iv) are immunologically unrelated to animal retrovirus strains; (v) possess an endogenous RNA-dependent DNA polymerase activity that can be banded at 1.16 g/ml in linear sucrose gradients. These results may be taken as suggestive evidence that HTD particles represent a novel group of unique retroviruses.

Properties of mouse leukemia viruses: VII. The major viral glycoprotein of Friend leukemia virus. Isolation and physicochemical properties

Abstract The major viral glycoprotein of Friend leukemia virus was isolated by a procedure comprising density gradient centrifugation, Con A affinity chromatography, and Sephadex gel filtration. It was obtained in milligram amounts and found to be pure by physicochemical and seroimmunological methods. It has a buoyant density in sucrose of 1.18 g/cm3 and a molecular weight of 71,000 as determined by SDS-polyacrylamide gel electrophoresis. A molecular weight of 58,000 was calculated from s20 = 4.05 S and D20 = 6.5 · 10−7 cm2/sec. The specific complement-fixing activity of the purified material was 50,000–100,000 complement-fixing units/mg protein.

Structure of simian virus 40 II. Symmetry and components of the virus particle

Abstract The symmetry of SV40 particles was examined and proved to be the T = 7 icosahedral surface lattice. Evidence is presented that the three types of shells of smaller size are derived from reassembled morphological units of normal particle shells. Examination of the surface structure of the smaller particles strongly suggested icosahedral surface lattices of T = 3 , T = 3, and T = 4 symmetry classes. Three different polypeptide chains with an average molecular weight of 16,350 ± 10% were identified in the protein moiety of the virus particle. The proportion was determined as 45.5 : 45.5 : 9. Four different approaches were used to determine the size of the virus DNA. A molecular weight of 2.25 × 10 6 ± 15% was established. A calculation of the particle weight from the size and number of the components yielded 17.3 × 10 6 ± 10%.

An electron microscopic study of bacteriophages from marine waters

The morphology of 75 bacteriophage strains isolated from water samples collected in the North Sea or in the northern Atlantic was studied by electron microscopy. Only tailed phages were observed (bradley groups A, B, and C). According to structural similarities, the strains are ascribed to 12 groups, 5 of which comprise types of marine phages not reported before. Four of these 5 groups include phage types that have not been detected from any other source. Among the phages isolated from northern Atlantic water a high incidence was observed of strains the particles of which have long appendages. Certain types of the northern Atlantic phages investigated were derived only from samples collected either east or west of the Azores. This finding agrees with former observations pointing to the existence of different populations of closely related bacteria east and west, respectively, of the northern Mid-Atlantic Ridge.

Transverse sectioning of plastic-embedded immunolabeled cryosections: morphology and permeability to protein A-colloidal gold complexes.

In order to provide data for meaningful interpretation and quantitation of immunogold labeling on cryosections their morphology and permeability to protein A-gold were evaluated: We studied plastic sections of immunogold-labeled ultrathin and semithick cryosections cut perpendicular to the original cryosection plane. Various soluble and insoluble antigens in different specimens (hemoglobin and histone H5 in chicken erythrocytes, tubulin in Leishmania cells, and outer membrane protein OmpA in Escherichia coli) were fixed with glutaraldehyde-formaldehyde, formaldehyde, or periodate-lysine-paraformaldehyde and incubated with specific antibodies and protein A-gold of different sizes. The cryosection surface may be rough or smooth depending both on the sectioned material and on dehydration and drying artifacts or possibly on the cutting process itself. Well-preserved sections are capable of withstanding considerable deformation without showing clefts or cracks. If the sectioned specimen is sufficiently fixed, protein A-gold is not able to enter the IgG-labeled sections significantly but follows surface irregularities. However, gold particles can be detected within visibly damaged sections.

C-type particles produced by a permanent cell line from a leukemic pig. II. Physical, chemical, and serological characterization of the particles.

Abstract The particle (PLCP) produced by a porcine lymphoma cell line is electron microscopically indistinguishable from C-type particles of other mammalian species possessing surface knobs, hexagonally arranged subunits in the core shell and a filamentous nucleoid. Furthermore, as in other C-type viruses, 60–70 S RNA as well as reverse transcriptase was found. Serologically PLCP could be clearly differentiated from murine as well as classical feline C viruses by the Ouchterlony test. Its mammalian C-type particle character was, however, documented by the presence of at least one interspecies antigenic determinant which is shared by most of the mammalian C viruses studied so far. According to its special interspecies reactivity, PLCP seems to be more closely related to woolly monkey (SSV-1), Gibbon ape, and RD 114 viruses than to classical feline and small rodents C viruses.

The Structure of Group II Adenoviruses

Summary Adenovirus types 13, 15, and 19 and their soluble antigens were studied by electron microscopy, applying the negative staining technique to purified material. Virus particles showed projections of 160 to 190 A length with a final knob arising from the 12 vertices of the icosahedron. Incomplete particles were also seen. The group-specific antigen consisted of free (‘hexon’) capsomeres. The type-specific antigen, which showed the phenomenon of haemagglutination in the presence of a heterologous immune serum, had the appearance of fibres with final knobs (‘fibre antigen’). The soluble haemagglutinin appeared as a star with a diameter of 450 to 600 A. It was composed of presumably 12 dumb-bell-shaped subunits, arranged in a regular fashion. The virus types studied were morphologically more closely related to type 3 (group I) than to type 5 (group III).

Properties of mouse leukemia viruses. IV. Hemagglutination assay and characterization of hemagglutinating surface components.

Abstract A hemagglutination (HA) assay for mouse leukemia virus (MuLV) concentrates is described. The assay gives linear dose-response data between concentrations of approximately 10 11 and 8 × 10 12 particles per milliliter. One HA unit was equivalent to approximately 5 sX 10 7 virus particles in those preparations where surface projections (knobs) were readily detectable on the virions. When knobs were removed during purification or by bromelain treatment, the HA activity was reduced or eliminated. Bromelain-treated virus also lacked glycoprotein component (s), did not absorb neutralizing MuLV antibody, and was noninfectious. Thus, the HA substructure is clearly a surface component of the virion and has an apparent association with the surface knobs, glycoprotein (s), and type-specific antigens. Treatment with neuraminidase and phospholipase C, as required for demonstration of HA activity, damaged the envelope of the virion but did not release hemagglutinating subunits or surface knobs. Active hemagglutinin recovered from enzyme-treated virus by degradation with Tween 80 and ether and density gradient centrifugation was stable under various physical treatments but was heat sensitive. Active hemagglutinin could also be recovered from non-enzyme-treated virus by similar procedures. Concentrated preparations of avian myeloblastosis virus and feline leukemia virus agglutinated chicken and sheep erythrocytes, respectively, but in both cases the specific HA activities were low and the reactions could not be inhibited by specific antisera.