E. Kellenberger

L'inclusion au polyester pour l'ultramicrotomie

Abstract The use of polyester, as embedding material for ultrathin sections, completely avoids swelling of the tissue fragments and formation of bubbles. We have found Vestopal W to be a very adequate commercial polyester, when used with adequate initiator (tert. butyl perbenzoate) and activator (cobalt naphthonate). The embedding procedure is similar to that used for other embedding materials. Acetone must be used, however, instead of ethanol for the dehydration. Even relatively thick polyester sections provide much higher resolution than methacrylate sections of comparable thickness. This fundamental difference can be ascribed to a different response of these embedding materials when bombarded with electrons: during decomposition and partial volatilization, methacrylate melts while Vestopal does not. As consequences of this melting, surface tension tends to compress the sectioned specimen, altering its spatial arrangement, while capillary effects blur the contours of its structural details. The specimens embedded in polyester, on the contrary, remain undistorted and clearly defined.

A new preparation method for dark-field electron microscopy of biomacromolecules

Alternating current glow discharge in vapors of amylamine renders carbon films suitable for electron microscope preparations of nucleic acids and proteins. The molecules are evenly distributed on the film and deposited with an efficiency of 10–20 %. Length measurements on naked, uncoated DNA (circular replicative form of fd and λ phages) demonstrate the reproducible results obtained with the method. Preliminary observations on RNA polymerase attached to DNA suggest that the internal structures of the protein may be observed and that the enzyme is probably attached asymmetrically to the DNA. The high contrast of dark-field microscopy permits resolution of biological structure at the limit of the microscope. Other factors, like background structures of the carbon film (microcrystallites) and destruction of the object, now become limiting factors.

Electron microscopical studies of phage multiplication: I. A method for quantitative analysis of particle suspensions

Abstract The agar-filtration method for preparing particle suspensions for the electron microscope is described and its application to particle counting is studied. The coefficient of variation of this method is found to be about 15%. The precision of the absolute determination of titers is, however, limited by the precision of the determination of the latex sphere diameter and is estimated to be 20 to 30%.

Functions and properties related to the tail fibers of bacteriophage T4

Abstract It is shown that adsorbability of T4 is regularly correlated with the extended state of the tail fibers, suggesting that in T4 fiber extension is a necessary condition for adsorption. Furthermore the extension and retraction of fibers is correlated with the dual sedimentation of T4 observed during ultracentrifugation. For T4, 38, which requires tryptophan for adsorption, electron microscopy shows the tail fibers to be extended in the presence of tryptophan and retracted in its absence. Phages with retracted fibers (no tryptophan) show a faster sedimentation than those with extended fibers (with tryptophan). In the absence of tryptophan T4, 38 does not show fiber extension even at pH 7, a condition sufficient for nontryptophan-requiring T4, whose fibers are retracted at pH 5. A conditional lethal mutation in gene 37 of phage T4D results in phages which lack tail fibers entirely. These phages no longer adsorb. In the ultracentrifuge, only the fast form can be observed whether at pH 7 or 5. Phages with extended fibers are more rapidly inactivated by ultrasonic waves than phages with retracted fibers. Measurements on electron micrographs show that the head size of T4 is invariant. A model of the functioning fiber apparatus is proposed and discussed.

Electron microscopical studies of phage multiplication. IV. The establishment of the DNA pool of vegetative phage and the maturation of phage particles.

Abstract Upon infection of Escherichia coli by phage T2, rapid disintegration of the bacterial nucleus is followed at 4 minutes by the formation of marginal vacuoles; after an additional 4 minutes a visible pool of phage-precursor DNA begins to develop. Even so early a step in this process as breakdown of the bacterial nucleus is inhibitable by chloramphenicol. Some 10 minutes after infection, phage DNA begins to condense into dense particles about the size and shape of phage, but lacking a protein membrane and hence extremely fragile, not surviving lysis in any recognizable form. The condensed particles then develop into an immature phage form, surrounded by an incomplete protein membrane which ruptures upon lysis, producing empty heads or doughnuts. The growth of the morphological DNA pool is unaffected by chloramphenicol when this is added 8 minutes or more after infection. No condensation of DNA occurs, however. Hence it is postulated that the condensation of DNA is produced by a yet unknown “condensation principle” of protein nature.

Electron Microscopical Studies of Phage Multiplication. II. Production of Phage-Related Structures during Multiplication of Phages T2 and T4.

Abstract During intracellular growth of T2 phage there is a production of rodshaped particles, which have the length of phage tail and which represent a structure related to the inner core of phage tail. They have little or no serum blocking power (SBP), but are to some extent adsorbable to bacteria. The intracellular appearance of rods and empty heads is studied in the presence and absence of proflavine. It is found that the production of both structures is not influenced by this substance, or by enrichment of the medium by amino acids. Proflavine depresses only the production of mature, active phage. The interpretation of these findings is discussed.

Studies on the morphopoiesis of the head of phage T-even: I. Morphological, immunological, and genetic characterization of polyheads

Conditional lethal mutations in gene 20 lead to the production of polyheads. Observed intracellularly in sections, polyheads are tubular structures. The cross sections are frequently pentagonal. Polyheads are filled with an unidentified internal substance which is different—at least in concentration—from the contents of normal phage. In a lysate most polyheads are open, but a few are sealed at one or both ends; most open polyheads appear empty upon examination. Serological studies show that polyheads and normal heads have at least one antigenic site in common. The normal heads have at least one more antigenic site than the polyheads. To produce polyheads, the normal functions of two “head” genes are necessary: (a) gene 23 which is believed to produce the protein subunit of the capsid, and (b) gene 31, the function of which has not yet been identified. A temperature-sensitive mutation L65 in gene 23 produces abnormal phage heads. This abnormality is also observed in polyheads produced by the double mutant tsL65 (in gene 23) -amN50 (in gene 20).

Studies on the morphopoiesis of the head of bacteriophage T-even: VIII. Multilayered polyheads

Abstract The major structural protein of the head of bacteriophage T4 can assemble to produce tubular forms. These tubular forms are particularly numerous in the case of the absence or inactivity of certain gene products which are necessary for the formation of the head shell or capsid. Multilayered tubular forms are observed intracellularly in mutants of gene 22. Upon lysis of the cells, the layers are found to separate into individual layers. The helical parameters of these tubes were studied by the optical diffraction method, using about 400 electron micrographs of negatively stained or shadowed tubes. The plane-lattice from which the helical net is derived has approximate hexagonal symmetry. The unit cell size is constant regardless of the diameters of tubes and is 110 ± 15A. The pitch angles of different tubes are, however, variable. The helical parameters of the innermost (i.e. narrowest) tubes are limited to a narrow range, whereas there is a wide distribution in the case of outer layers. By means of optical diffraction of shadowed specimens, which have one-sided images, the helical lattice of the tubes was found to be right-handed. The inner diameter of the innermost layer is constant (400 ± 20A) and independent of the number of supplementary layers. The successive layers are 94Athick and appear to fit tightly around each other. The bearing which these and other observations have on the mechanisms of the inheritance of form is discussed. Each layer seems to act as a form-determining core for the following one.

On the fine structure of normal and “Polymerized” tail sheath of phage T4

“Polysheaths’ are frequently found in lysates of phage T4. We present evidence that they are aberrant assemblies of tail material in a structure identical with contracted sheath. Polysheath is found in a “smooth” and a “helical” form. From our observations we deduce that polysheath is composed of several helical bands with a pitch of 30 degrees. The full set of helical bands leads to the smooth form; in the helical forms some of the bands are lacking. To these observations on polysheath, we add observations on contracted and extended sheath, which are mostly confirmations of previous results. Taken together they allow us to propose a model for the sheath structure and its transition from the extended to the contracted form. We postulate that the subunits exist in two forms (A and B) different in the internal conformation of the polypeptide chain. Each conformation is assorted with a different set of superficially available chemical binding sites. According to the available set, the subunits would either settle on the central tail tube to form the close-packed array of the extended sheath (A) or produce free polysheath by self-assembly (B). Contraction would be initiated by the transition of each subunit from form A into form B; the binding of the subunits to the central core would be released and the subunits be rearranged according to this second set of binding sites. Our model can be further refined when the conformational change is explained by an allosteric effect.

On the fragility of phage T4-related particles☆

Abstract By means of three different methods of specimen preparation for the electron microscope and two methods of in vitro complementation, each involving different degrees of physical stress, the fragility of normal, tailless heads and of immature or abnormal heads produced by mutants in the X genes was estimated. Stability against action of DNase was checked. Only genes 16 and 17 were found to be possible candidates for producing either empty shells, which are filled later or highly fragile, already filled heads. The heads made by mutants in gene 49 are always full, although in a somewhat abnormal fashion. It is shown that genes 64, 50, 65, 4 are probably involved in tail-head junction because in these mutants the attachment of the tails is particularly fragile.