Electron Microscope Study of DNA-Containing Plasms

Kellenberger, E.; Ryter, A; Séchaud, Janine

doi:10.1083/jcb.4.6.671

Cited by 976 publications

(144 citation statements)

References 31 publications

(23 reference statements)

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“…The osmium tetroxide fixation and polyester (vestopal) embedding method described by Kellenberger et al (9) was used without modification on Supported by a grant from the National Research Council of Canada. most samples.…”

Section: Methodsmentioning

confidence: 99%

“…Although it was presumed that both surfaces of this spore septum might be covered by reflections of the cytoplasmic membrane, these could not be resolved in methacrylate embeddings. The process of sporulation has now been reinvestigated, using osmium fixation and polyester embedding procedure developed by Kellenberger and associates (9) since this procedure has proved useful in resolving cell layers (8). The result so obtained presented a more complex picture of the mechanism of forespore formation than that indicated earlier and thus prompted a comparative study of this division with that typical of vegetative growth.…”

Section: Introductionmentioning

confidence: 99%

“…9 was also drawn with this in mind). After uranyl acetate treatment (9), the borders of the unit plasma membrano (those marked ? in Fig.…”

Section: Development Of the Forespore In Bacillimentioning

confidence: 99%

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Participation of the Cytoplasmic Membrane in the Growth and Spore Formation of Bacilli

Fitz-James

1960

The Journal of Cell Biology

291

115

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A polyester embedding technique was used to study the early stages of spore formation in members of the genus Bacillus in order to investigate further the origin and nature of the initial spore septum and the resulting forespore envelope. Whereas previously, with a methacrylate procedure, this layer had appeared to be continuous with the cell wall, this study reveals it as a double layer of cytoplasmic membrane. Perisporal, membranous organelles connected both to the developing forspore envelope and to the cytoplasmic membrane were encountered in the four species studied. Similar organelles were prominent during growth at the sites of transverse septa formation. These were connected to, or continuous with, the cytoplasmic membrane and often adherent to the chromatin bodies of the dividing bacilli.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Participation of the Cytoplasmic Membrane in the Growth and Spore Formation of Bacilli

Fitz-James

1960

The Journal of Cell Biology

291

115

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“…In addition, molecules are not visible when their long dimension is parallel to the direction of shadowing. More recently it has been shown that contrast of nucleic acid containing structures can be enhanced in situ by staining with heavy metal salts (12,14,26,27), and work with tissue sections, bacteria, viruses, and subcellular particles has shown that the nucleic acid itself is mainly stained in these structures (12)(13)(14)(15)24). Our own work with heavy metal staining of small viruses has convinced us that it should be possible to render single molecules of nucleic acids clearly visible in the microscope, and the results obtained so far are reported in this paper.…”

mentioning

confidence: 99%

Electron Microscopy of Dna Molecules "Stained" With Heavy Metal Salts

Stoeckenius

1961

The Journal of Cell Biology

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Single DNA molecules can be rendered visible in the electron microscope by "staining" with water-soluble salts of heavy metals. The best results were obtained with lanthanum nitrate, uranyl acetate, and lead perchlorate. The molecules appear as filaments approximately 20 A wide. Their length was not determined, but it could be shown that it varied with the molecular weight of the DNA used. The same heavy metal salts will preferentially "stain" the nucleic acid in a protein-DNA complex. Evidence is provided for the possibility of a partial separation of a double-stranded molecule into single strands on adsorption to the supporting film.Mounting interest in the role of nucleic acids in biology and increasing knowledge of their molecular structure have prompted numerous attempts to supplement the information gained through indirect methods by direct observation of single molecules in the electron microscope. Several techniques have been used to obtain a suitable distribution of single molecules and to increase contrast in the preparation. After drying from a drop of dilute solution on a supporting film of collodion, Formvar, or carbon, DNA is usually found in large aggregates, in which little detail is visible. Spraying the solution from an atomizer onto a hydrophilic surface will result in a more homogeneous distribution of the DNA over a larger area and will allow the visualization of single molecules. Hall (10) and Hall and Litt (11) used the surface of freshly cleaved mica, which has the additional advantage of providing a very smooth background for subsequent shadowing, but transfer of the preparation to a grid presents some difficulties. Birbeck and Stacey (6) obtained satisfactory results by using a polymer with positively charged groups as the supporting film, so that DNA could be sprayed directly on the grid. Neither method, however, seems to be well suited for work with material of the very high molecular weight found in native DNA from many sources.A new technique recently published by Beer (4, 5) offers definite advantages in this respect. By streaking a film-covered grid over the surface of a DNA solution and by carefully draining off, in the same direction, the surplus adhering to the grid, he succeeded in attaching to the film single molecules in the form of nearly straight and parallel strands.Still another interesting approach is that of Kleinschmidt and Zahn (16,17), who spread DNA in a monomolecular film of protein on a water surface. The protein is necessary since DNA alone will not form a film, and the protein, furthermore, keeps the molecules of DNA separated so that they can be individually resolved.In all these investigations shadowing with heavy metals has been used to render the nucleic acid visible in the microscope. This procedure, however, limits the resolution to such a degree that it is difficult if not impossible to distinguish clearly a single molecule from two molecules in close contact. In addition, molecules are not visible when their long dimension is parallel to the

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“…Cell morphology was determined with a Leitz phase contrast microscope. Thin sections were prepared for electron microscopy by methods previously described (Kellenberger et al, 1958;Kennedy et al, 1975). Cell dimensions were determined from both phase contrast and scanning electron photomicrographs (Merkel et af., 1978).…”

Section: Thermus Aquaticus Yt-1 (Atcc 25104)mentioning

confidence: 99%

Isolation and Peptidoglycan of Gram-negative Hydrocarbon-utilizing Thermophilic Bacteria

Merkel¹,

Stapleton²,

Perry³

1978

Journal of General Microbiology

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141Four Gram-negative, non-sporulating, aerobic, obligate thermophilic bacteria, isolated from non-thermal environments by enrichment with n-heptadecane as substrate, utilized n-alkanes, carbohydrates and organic acids as sole source of carbon and energy and also grew on complex media. The growth rate of these organisms, when utilizing n-heptadecane as substrate, was markedly increased by adding a low concentration (7.5 mg 1-l) of yeast extract. They grew optimally between 55 and 65 "C, and at a pH between 6.2 and 7.5. The mol yo G + C for all was between 5 1 and 58. On the basis of the amino acid and amino sugar compositions of their peptidoglycan, these organisms and other Gram-negative thermophilic bacteria can be divided into four distinct groups. Group A includes the newly isolated hydrocarbon-utilizing bacteria which have nearly equimolar amounts of glutamic acid, alanine, diaminopimelic acid and glucosamine. Group B consists of obligate hydrocarbonutilizing microbes that have lower molar ratios of glutamic acid and diaminopimelic acid, and contain either ornithine or lysine. The previously isolated non-hydrocarbon-utilizing therinophiles (K-2, Thermus aquaticus YT-1, Thermus X-1) and a newly isolated organism from a hot spring comprise group C and contain glycine, ornithine, no diaminopimelic acid, and much lower molar ratios of glutamic and muramic acids than in groups A and B. Thmnowicrobiurn roseurn lacked peptidoglycan and is placed separately in group D.

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Electron Microscope Study of DNA-Containing Plasms

Cited by 976 publications

References 31 publications

Participation of the Cytoplasmic Membrane in the Growth and Spore Formation of Bacilli

Participation of the Cytoplasmic Membrane in the Growth and Spore Formation of Bacilli

Electron Microscopy of Dna Molecules "Stained" With Heavy Metal Salts

Isolation and Peptidoglycan of Gram-negative Hydrocarbon-utilizing Thermophilic Bacteria

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