Spectrin-actin associations studied by electron microscopy of shadowed preparations

By shadowing specimens dried onto mica sheets we have obtained clear images of actin crosslinked by spectrin, an actin-binding protein found in erythrocytes. We conclude that spectrin dimers possess a single binding site for F actin. Tetramers formed by head-to-head association of two dimers possess two actin binding sites, one at each tail. Polymerizing G actin in the presence of spectrin tetramers or mixing preformed F actin with spectrin tetramer plus band 4.1 results in an extensively crosslinked network of actin filaments. When G actin is polymerized in the presence of spectrin at spectrin:actin mole ratios close to that present on the erythrocyte membrane, large amorphous protein networks are formed. These networks are clusters of spectrin around 25 nm diameter structures which may be actin protofilaments. These networks are similar to the cytoskeletal network seen after erythrocyte membranes are extracted with detergent, and may represent the first in vitro assembly of a cytoskeletal complex resembling that of the native cell both biochemically and structurally.     

Mapping restriction sites on large DNAs by electron microscopy

We have developed a novel technique to map restriction sites on large duplex DNAs by electron microscopy. In this method, the sample DNA is first cut with a restriction enzyme. The resulting fragments are briefly digested with Escherichia coli exonuclease III, and treated with wheat germ RNA polymerase II to fill-in with RNA the resulting gaps. These small RNAs, complementary to sequences immediately adjacent to either side of the restriction site, are isolated from the DNA template and R-looped to the full-length DNA. When this material is prepared by the formamide-cytochrome spreading technique, small bubbles are visible wherever there is a restriction site on the DNA. Improved methods of mapping are outlined.     

Scanning electron microscopy of experimental adiaspiromycosis

Adiaspiromycotic granulomas of mice experimentally inoculated with fungusEmmonsia crescens Emmons et Jellison 1960 were examined by scanning electron microscopy. Their morphology, surface structures, and germinating adiaspores isolated from granulomas are described.     

Mapping and length measurements of restriction enzyme fragments by electron microscopy.

1. We have mapped by electron microscopy the DNA-fragments formed by the action of the restriction endonuclease from Arthrobacter luteus of phi X 174 replicative form DNA. These fragments were separated by polyacrylamide gel electrophoresis and hybridized to phiX 174 single stranded DNA. The partial duplex molecules were inspected in the electron microscope. In this way the relative order of eleven fragments ranging in size from approximately 100 to 1000 nucleotide pairs has been established and compared with that deduced from reciprocal digestion studies. 2. The measured lengths of the fragments agreed well with the lengths found by gel electrophoresis. 3. The purity of the isolated fragments was checked. Most of the contaminating fragments derive from nearest neighbours in the preparative polyacrylamide gels.     

Mapping of mitochondrial 4 S RNA genes in Xenopus laevis by electron microscopy

The distribution of sites hybridizing with mitochondrial 4 S RNA molecules on mitochondrial DNA of Xenopus laevis has been mapped in relation to the ribosomal RNA genes and EcoRI restriction endonuclease sites. RNA molecules linked to ferritin were employed for this purpose. We have obtained evidence for 15 4 S RNA sites on the H-strand and six sites on the L-strand of X. laevis mtDNA^∥. An indication of the possible existence of one additional site on the H-strand and four additional sites on the L-strand has been obtained. One 4 S RNA site is located in the gap between the two rRNA genes, and one site flanks each outside end of the rRNA genes. The other 4 S RNA sites are distributed almost evenly throughout both strands of the mtDNA. A comparison with the map of 4 S RNA sites on the mtDNA of HeLa cells (Angerer et al., 1976) suggests considerable evolutionary conservation of site organization.     

X-ray diffraction and electron microscopy studies of frozen erythrocyte membrane preparations

Well-defined X-ray diffraction patterns have been recorded from erythrocyte membranes in the frozen state. At ?40°C, lamellar periodicities range from 19 to 95 nm depending on the glycerol content (0–40%, respectively). Freeze-fracture electron micrographs of samples frozen in two stages to approximate to the diffraction conditions show ice formation external to membrane stacks. The membrane stacks have periodicities of the same order of magnitude as those obtained by X-ray diffraction.     

Transmission electron microscopy of tissue prepared for scanning electron microscopy by ethanol-cryofracturing.

Tissue processed for scanning electron microscopy by ethanol-cryofracturing combined with critical point drying was embedded and sectioned for transmission electron microscopy. Study of specimens cut in a plane passing through the fracture edge indicated that preservation of cellular fine structure of fractured cells was excellent. Even at the most peripheral edge of the fracture there was no evidence that movement of cytoplasmic components occurred to distort the original structural organization of fractured cells. Lack of cytoplasmic detail in ethanol-cryofractographs has been due more to the nature of the fracturing of the tissue and to the obscuring effects of the metal coating than to structural deformation at the fracture edge or to limitations in resolving power of the scanning electron microscope used.