Ronald B. Luftig

An accurate measurement of the catalase crystal period and its use as an internal marker for electron microscopy.

A Siemens Elmiskop I microscope operated under optimum stability conditions was calibrated by use of the 12.4 A spacing of copper phthalocyanin crystals. The latter period was determined from the corresponding electron diffraction pattern. Extensive measurements were then made for the line spacing of negatively stained catalase crystals at magnifications of 45,000–65,000 ×, and a period of 88 ± 3 A was obtained. To illustrate the efficacy of catalase as an internal magnification standard for electron microscopy, the sizes of bacteriophage T7, cyanophage LPP-1, and ferritin were determined within a 3% error. These particles were found to have edge-edge distances of 626 ± 18 A, 615 ± 19 A, and 111 ± 3 A, respectively. A further test with catalase crystals as an internal standard was the ability to distinguish by electron microscopy the pH induced isomorphic swelling of bromegrass mosaic virus whose diameter increased from 271 ± 11 A at pH 6.1 to 299 ± 13 A at pH 7.5.

Localization of RNA tumor virus polypeptides: I. Isolation of further virus substructures

Abstract The internal components of avian myeloblastosis virus (AMV) and Friend murine leukemia virus (FLV) were isolated and characterized. AMV cores contained three of the five major virus polypeptides having molecular weights of 27,000, 15,000, and 12,000. FLV cores consisted of three of the four major components, and these have molecular weights of 31,000, 15,000, and 10,000. For both types of cores the 15,000 dalton component was present in least amounts. The internal ribonucleoprotein was isolated from each of the cores and shown to consist of the most basic protein of the respective agents. The properties of the proteins and their location in the virions indicated a strong similarity between the avian and mammalian agents.

An ultrastructural study of virions and cores of reovirus type 3

Abstract The results of an ultrastructural examination of virions and cores of reovirus type 3 are reported. The diameter of virions is 764 ± 43 A, that of cores 521 ± 31 A. The capsomers of both the outer an the inner shell (the core) are so arranged that 20 appear to be located peripherally. Those comprising the outer shell are 90 A in diameter and 10 A apart, those of the inner shell are 40 A in diameter. Both seem to be uniform in size and shape. Rare head-on views of both confirm these size estimates and also suggest that both are hollow truncated pyramids. Spatial interrelationships between neighboring capsomers on neither outer nor inner shells could be discerned clearly enough to permit assignation of any particular symmetry pattern, not even when virions were treated briefly with chymotrypsin, which enhances surface detail. The total number of capsomers in each shell follows directly from the fact that 20 are located on the periphery: the maximum number is 127. This is substantially in excess of the number (92) required for a model proposed by others which, in addition, requires 18 peripherally located capsomers. Another model, which postulates 180 capsomers located around 92 holes, is also not compatible with our results. Cores possess 12 projections or spikes located on 5-fold vertices, demonstrating that at least these reovirion components are arranged according to icosahedral symmetry principles. Their diameter is 100 A, and they project about half way to the outer surface of the virion. They appear to be hollow at their distal end. It is conceivable that they are the organelles through which transcripts of reovirus genome RNA are released. Dissolution of cores with sodium dodecyl sulfate releases viral RNA as a tightly associated mass of strands which appear to be coiled either randomly or concentrically. These results are discussed with reference to the structure of the reovirion capsid shells, and the possible structure and function of the core spikes.

An electron microscope study of Rauscher leukemia virus

Abstract The effect of various stains on the morphology of Rauscher leukemia virus was examined. The virus appeared as a round, membrane-bound structure when prefixed with glutaraldehyde or formalin and stained with uranyl acetate (UA). Other stains, such as sodium phosphotungstate, sodium silicotungstate, or ammonium molybdate caused disruption of over 80% of the virus particles. They then appeared as smaller, tailed particles or swollen, heavily stained membranous structures. In the prefixed, UA-stained preparations, the particles were accurately measured using the catalase crystal internal marker technique. The intact virus had an average diameter of 1470 A. Also present were smaller particles with an outer diameter of 1090 A, which occurred at a level of 20–30%. The proportion of these smaller structures could be increased to 70% of the total particles by a brief treatment with 0.01% dodecyl sodium sulfate (SDS). This suggests that the outer envelope of the intact virus was selectively removed, thus exposing an internal nucleoid. This is consistent with the observation that isolated viral outer envelopes measured 200 A across. The nucleoids obtained by SDS-treatment banded in cesium chloride (CsCl) at an average density of 1.226 g/ml, compared to the value of 1.18 g/ml for the fully enveloped virion, which also suggests the loss of a lipid envelope. Occasionally, nucleoid particles were fortuitously stained to reveal an internal single or double-stranded whorled substructure. The double-stranded form was 2.5 times more prevalent in virus harvested at 3 hr than at 24 hr. The contour length of both whorled forms was about the same and corresponds to a three-dimensional coil 1.1 – 1.5 μ long. Upon prolonged treatment of virus harvested at 3 hr with 0.01% SDS, the whorled structures could be liberated from the nucleoids. They appeared as strands about 1.18 μ long, and banded in CsCl at an average density of 1.343 g/ml. Their localization in the particle by UA staining, as well as their density suggest that these strands are nucleoprotein in nature. Models based on how the strand is packaged in the nucleoid are suggested to explain how the various internal forms could arise in the virus population.

Comparison of blue-green algae virus LPP-1 and the morphologically related viruses GIII and coliphage T7☆

Abstract The blue-green algae viruses LPP-1 and G III , and coliphage T7 are morphologically similar in that they each possess hexagonal capsids, about 600 A across, with a short tail, 150–200 A in length by 150 A in diameter, attached to one of the vertices. The blue-green algae viruses LPP-1 and G III appear to be identical, since they have the same inactivation kinetics with LPP-1 antiserum. LPP-1 and T7, however, are serologically unrelated. They appear to be genetically unrelated as well, since LPP-1 DNA hybridizes to an insignificant extent with RNA synthesized in vitro on a T7 DNA template, despite a similarity in average G-C content of the two DNAs.

Studies on the structure of blue-green algae virus LPP-1☆

Abstract Blue-green algae virus LPP-1 has a head capsid hexagonal in projection, with edge-to-edge distance of 600 ± 20 A, and a short tail 200 A long and 150 A in diameter attached to one of the head capsid vertices. These observations made on intact virus particles have been extended by electron microscopic and preliminary chemical characterization of separated heads and tails. Purified heads dissociated in 6 M urea show a single band on acrylamide gel electrophoresis at pH 9; this component of the head protein elutes from a calibrated G-100 Sephadex column in a volume corresponding to a molecular weight of 17,500. Since the molecular weight of the total head protein is 24–28 × 10 6 , the head is comprised of 1400–1600 structure units. Purified tail preparations contain at least three distinguishable particles: cylinders 200 A long × 105 A in diameter, capped cylinders 300 A long, and joined cylinders 400–450 A long. These probably correspond to tails without capitals, tails with capitals, and tail dimers, respectively. End views of the tail include pinwheels on which six elements are clearly resolved.

Morphological, Chemical, and Antigenic Organization of Mammalian C-Type Viruses

New features in the architecture of mammalian type C viruses, in particular knoblike surface projections and hexagonally arranged subunits on the core shell could be demonstrated by electron microscopy, taking advantage of newly developed preparation techniques. As examples, murine leukemia viruses (MuLVs) and newly isolated porcine and bovine C viruses are presented. The major proteins of a MuLV were isolated and partially characterized in chemical terms and with respect to their serological and other biological activities, such as interfering and hemagglutinating (HA) capacity. Most of the characterized proteins could be localized in particular substructures of the virion either by selective removal or isolation of electron microscopically identifiable constituents. The information obtained allowed the design of a more detailed model of mammalian C viruses. Special attention was devoted to the further characterization of interspecies antigens of mammalian C viruses. Different antigenic determinants were revealed. Their distribution allows further subgrouping of mammalian C viruses.

DNA Polymerases and a Possible Multi-Enzyme Complex for DNA Biosynthesis in Eukaryotes

During the past decade considerable progress has been made toward understanding the mechanism and control of DNA replication. The greatest advances have come in studies of prokaryotic DNA replication and these results have facilitated studies of eukaryotic DNA replication. The points emerging are that DNA replication in prokaryotes and eukaryotes involves the concerted action of several enzymes, non-enzymic factors and an integrated intracellular arrangement.

Bacteriophage T4 head morphogenesis. V. The role of DNA synthesis in maturation of an intermediate in head assembly.

Abstract On the basis of metabolic-inhibitor studies it was found that maturation of T4 head intermediates, which had accumulated in tsC9 (gene 49) mutant infected cells, was apparently dependent on continued DNA synthesis. This requirement for DNA synthesis in head maturation was confirmed by using a gene 43 mutant (tsP36) blocked in the enzyme, T4 DNA polymerase. In temperature shift-up experiments with tsP36-infected cells it was found as previously reported ( Luftig and Ganz, 1972 ) that 300s particles accumulated when enzyme synthesis was halted. These latter structures appear identical to the gene 49-defective intermediates since, when isolated, they both: (i) had an average sedimentation coefficient of 310 ± 20s and a nucleoprotein density of 1.34 g/cc; (ii) had the major capsid polypeptide in a cleaved state; (iii) were quantitatively rescued (to a >50% level in 40 min) after the mutant-blocked function was recovered, and (iv) showed a reduced ability to convert to phage when an inhibitor of DNA synthesis such as fluorodeoxyuridine (FUdR) was added at the time the function was rescued. The maturation of rapidly labeled 300s wild-type (T4D+) head intermediates was also found to be sensitive to inhibitors of DNA synthesis. Upon isolation these heads had the same properties as the above gene 49 and gene 43 mutant-defective intermediates. This strongly suggests that DNA synthesis is a normal requirement for maturation of a T4 capsid intermediate, whose properties are defined above.