E. Boy de la Tour

Metaphase chromosome structure. Involvement of topoisomerase II.

SCI is a prominent, 170,000 Mr, non-histone protein of HeLa metaphase chromosomes. This protein binds DNA and was previously identified as one of the major structural components of the residual scaffold structure obtained by differential protein extraction from isolated chromosomes. The metaphase scaffold maintains chromosomal DNA in an organized, looped conformation. We have prepared a polyclonal antibody against the SC1 protein. Immunolocalization studies by both fluorescence and electron microscopy allowed identification of the scaffold structure in gently expanded chromosomes. The micrographs show an immunopositive reaction going through the kinetochore along a central, axial region that extends the length of each chromatid. Some micrographs of histone-depleted chromosomes provide evidence of the substructural organization of the scaffold; the scaffold appears to consist of an assembly of foci, which in places form a zig-zag or coiled arrangement. We present several lines of evidence that establish the identity of SC1 as topoisomerase II. Considering the enzymic nature of this protein, it is remarkable that it represents 1% to 2% of the total mitotic chromosomal protein. About 60% to 80% of topoisomerase II partitions into the scaffold structure as prepared from isolated chromosomes, and we find approximately three copies per average 70,000-base loop. This supports the proposed structural role of the scaffold in the organization of the mitotic chromosome. The dual enzymic and apparent structural function of topoisomerase II (SC1) and its location at or near the base of chromatin loops allows speculation as to its involvement in the long-range control of chromatin structure.

The metaphase scaffold is helically folded: Sister chromatids have predominantly opposite helical handedness

We have studied the three-dimensional folding of the scaffolding in histone H1-depleted chromosomes by immunofluorescence with an antibody specific for topoisomerase II. Two different types of decondensed chromosomes are observed. The majority of the chromosomes are expanded, and the central fluorescence signal is surrounded by a large halo of chromatin. A much smaller number of chromosomes are more compact in length; they contain a smaller halo of chromatin and their scaffolds are not extended but folded into a genuine, quite regular helical coil. This conclusion is based on a three-dimensional structural analysis by optical sectioning. The number of helical coils is related to chromosome length. Surprisingly, sister chromatids have predominantly opposite helical handedness; that is, they are related by mirror symmetry.

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.

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.

Involvement of IS1 in the dissociation of the r-determinant and RTF components of the plasmid R100.1.

SummaryThe formation of the r-determinant pLCl and of the RTF pAR132 from the composite plasmid R100.1 was investigated. The general location of IS1 sequences on the three plasmids was established by hybridization of λr14 CII::IS1 DNA to EcoRI generated fragments of the various plasmids separated by agarose gel electrophoresis and transferred directly to nitrocellulose filters. The position of IS1 sequences on these fragments and the homologies between fragments were analyzed by electron microscopy of heteroduplex molecules. The results show that the excision of both pLCl and pAR132 occurred by an exchange between the two IS1 sequences present on R100.1.

Studies on the morphopoiesis of the head of phage T-even: II. Observations on the fine structure of polyheads

Electron micrographs of flattened polyheads frequently show moire patterns of a hexagonal type. These are due to an angular displacement of the upper surface with respect to the lower, which is the necessary corollary of a helical structure of polyheads. By our observations we confirm the conclusions reached by use of another method by Finch et al. (7) in that the surface lattice of the originally tubular polyheads is of a hexagonal (P6) design. We determine the unit lattice vector to be of 67.4 ± 1.6 A. From the periodicity of the resulting moire pattern the angle formed by the upper and lower lattice can be calculated and is found to differ according to the polyhead, indicating different pitches of the helix. The circumference is constant along one individual tube but varies from polyhead to polyhead within a factor of about two. On polyheads we occasionally observe distinct morphological subunits (“capsomeres”) which appear to be segments of a hollow cylinder with an outer diameter of 70–80 A and with a hole of 20–40 A. On normal capsids of T4, capsomeres are not readily visible. Polyheads and capsids are also different with respect to buoyant densities and chemical stability.

Tn10 mediated integration of the plasmid R100.1 into the bacterial chromosome: inverse transposition.

SummaryUpon integration into the bacterial chromosome the drug resistance plasmid R100.1 often loses its tetracycline resistance character. We have analyzed an Hfr strain formed by such an integration and an R-prime plasmid derived from it. We find that integration took place within the Tn10 transposon, that the two IS10 sequences were retained, but that at least 80% of the transposon segment located between them, and carrying the tetracycline resistance genes, had been lost. We suggest that integration of R100.1 was mediated by an inverse transposition using the IS10 sequences.

Some properties of the chloramphenicol resistance transposon Tn9

SummaryWe have isolated variants of the plasmid RTF which have received the transposon Tn9 from bacteriophage P1Cm. We have shown by the formation of heteroduplex molecules between one RTF: Tn9 derivative and R100.1 that Tn9 is homologous to the r-determinant region of R100.1 which carries the determinants for chloramphenicol resistance. This suggests that Tn9 was derived from an r-det like structure by deletion, possibly mediated by one of the flanking IS1 elements. In spite of the similarity in structure between Tn9 and r-det however, we have demonstrated two distinct differences in the behavior of these two elements: 1) Tn9 but nor r-det, is able to amplify, by a recA dependent mechanism, when cells harboring RTF::Tn9 are grown in the presence of chloramphenicol, and 2) Tn9, unlike r-det, does not form extrachromosomal circular molecules when RTF::Tn9 is tegrated into the bacterial chromosome.