50 years ago in microscopy

Editor's note: Citation Classics Commentaries were written by the authors of studies that were some of the most highly cited papers between 1961 and 1975. The essays were originally published between 1977 and 1993 in Current Contents, a publication of the Institute for Scientific Information (ISI), now Thomson Scientific. (ISI was founded by Eugene Garfield, also the founder of The Scientist.) In this essay, published in 1978, a biochemist and microscopist describes his 1958 paper describing a new technique for preparing thin sections for the electron microscope.The study, led by Eduard Kellenberger, then at the University of Geneva, made an impact more for its advancements of microscopy than for its biological findings regarding DNA. In the past, researchers had fixed tissue for electron microscopy with a type of plastic called methacrylate, which could create artifacts in the tissue, explained Bruno Strasser, a science historian at Yale. Kellenberger's study defined a new set of fixation conditions using OsO4 that became standard in electron microscopy. In the 1970s, Kellenberger went on to develop new embedding media called Lowicryl resins, which replaced the osmium method and are still used today.Kellenberger's study was cited 915 times between 1961 and 1980, and more than 1300 times in total. Kellengerger's coauthor Antoinette Ryter, then a biochemistry student at the University of Geneva, later joined the Institut Pasteur, and after receiving her doctorate, directed the institute's electron microscopy lab until she retired in 1989. She lives in Issy-les-Moulineaux, France. Janine Séchaud, a physicist and the third author on the study, remained at the University of Geneva until she retired in 1995. She still lives in Geneva. Kellenberger moved to the University of Basel in 1970, where he helped form the Biozentrum, an interdisciplinary research institute. He retired in 1990 and died in 2004 of heart failure.ºººººThe initial idea of a series of papers -- of which this was the second -- was to establish a test for distinguishing condensed DNAplasms of eukaryotes, e.g., metaphase chromosomes from metabolically active, noncondensed DNA, e.g., of DNA-plasms in the interphase of eukaryotes or during all times in prokaryotes. At that time the possibility was still open that the histones condense the DNA and render it thus metabolically inert. By removal of histones a decondensed DNA-plasm would arise which would be metabolically active. The decondensed DNA-plasms of bacterial nuclei and vegetative phage were known to be continuously replicating and transcribing. Some experiments then suggested that bacteria have no histones and in general fewer proteins bound to their DNA. My working hypothesis at that time was thus that during interphase the DNA-plasm of eukaryotes might be organized similar to that of prokaryotes; only the condensed metaphase chromosomes would have been specific for eukaryotes. The later applications of the methodology of this paper by others and ourselves showed that the DNA-plasms of mitochondria, chloroplasts, blue-green algae, dinoflagellates, and kinetoplasts of trypanosoma had a structure and behavior in fixation (coagulation-sensitive) analogous to that of bacteria. The DNA-plasm of interphase nuclei of eukaryotes, however, turned out not to be coagulation-sensitive. It also did not have the same fibrillar aspect. This result was suspected to be in relation to DNA binding proteins but only with our present knowledge can we understand it: in eukaryotes most of the histones always stay associated with the DNA of the chromatin. For bacteria, DNA binding proteins (different from histones) are demonstrated, but they are still found only in amounts which lead to much smaller protein-DNA ratios as in eukaryotes. Hence, it would be understandable that prokaryotic DNA-plasms behave similarly to naked DNA, while chromatin does not.Although we intended to draw the attention of the reader to the different organizational states of the nuclear material and how it can efficiently be investigated by electron microscopy of thin sections, I believe that the paper is mostly quoted as reference for a certain condition of fixation, which is mainly the result of an enormous effort of my collaborator Antoinette Ryter, then technician, now research director at the Pasteur Institute in Paris. It is only now, after the nucleosomes have been described, that some people are becoming aware again of possible differences between the prokaryotic and the eukaryotic type of organization of the nuclear material Others do not like differences and seize anything which could appear as evidence that bacterial DNA is organized with the same 'beads' as is the chromatin of eukaryotes. Has it not been demonstrated that SV40 virus is constructed by way of nucleosomes? As for most people viruses are lower organisms than bacteria, do we not have to expect nucleosomes also in bacteria? This wishful thinking reminds me of earlier times, when it became clear that microorganisms have genetics and when people thus tried very hard to demonstrate spindles and centrosomes in bacteria! Hopefully, further thorough investigation of bacterial chromosomes will not be inhibited by preconceptions and soon an explanation of the above discussed fundamental difference of prokaryotic and eukaryotic DNA-plasms will be provided.-Eduard Kellenberger, Biozentrum der Universität Basel, Switzerland, July 24, 1978Reference: E. Kellenberger, A. Ryter and J. Séchaud, "Electron microscope study of DNA-containing plasms. II. Vegetative and mature phage DNA as compared with normal bacterial nucleoids in different physiological states," 1958. J. Biophys. Biochem. Cytol., 4:671-678.

50 years ago in microscopy

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