Rewriting the Book on Nucleic Acids

Every cultured person today has heard of DNA, RNA and protein synthesis. But nucleic acids were not so well-known in 1927, when Albert Dalcq asked me to study the localization of "thymonucleic acid" in ovarian eggs (ovocytes). Biochemistry textbooks merely said that there are two kinds of nucleic acids, "animal" and "vegetal." Thymonucleic acid, a typical animal nucleic acid, had a queer sugar residue, which makes it a DNA. Plant nucleic acids, like zymonucleic acid from yeast, contained a pentose residue, a characteristic of our RNAs. Both nucleic acids, as indicated by their names, were located in the cell nuclei—DNA in animal cells, RNA in plant cells. Their function was unknown, but one could exclude a genetic role in view of their low molecular weight. Chemical embryologists displayed limited interest in nucleic acids: Joseph Needham devoted only 14 of 1,724 pages to them in his definitive Chemical Embryology (1931). R. Feulgen had found in 1924 that mildly hydrolyzed DNA gives a classical aldehyde color reaction, forming a violet compound with fuchsine sulfurous acid. He applied this reaction (the Feulgen reaction) to histological sections and discovered that all nuclei (animal and plant) stained violet. He concluded correctly that DNA is present in plant as well as animal cell nuclei. But biochemists, who viewed cytochemistry with contempt, did not believe in Feulgen's results and still contended that plant nuclei contained RNA and not DNA. My own work with the Feulgen reaction convinced me that, contrary to common belief, the slender ovocyte chromosomes contain DNA throughout ovogenesis; it also showed a negative reaction in the egg cytoplasm. Since DNA is a constant constituent of the egg chromosomes, I thought it might play a genetic role. Feulgen-stained eggs showed no cytoplasmic staining, which ruled out the existence of a large DNA reserve in the egg cytoplasm. Such a DNA pool was required by E. Godllewski's "migration" theory: on the basis of morphological studies on eggs stained using conventional methods, he held that there is no DNA synthesis when eggs divide into many cells, but rather a migration of a preexisting cytoplasmic DNA into the daughter nuclei. In sharp contrast with Godlewski's idea stood J. Loeb's "total synthesis" theory that DNA would be synthesized de novo during embryonic development at the expense of small precursor molecules. I spent two summers (1931 and '32) at the Oceanography and Marine Biology Research Center in Roscoff, France, studying DNA synthesis in sea urchin embryos. I met there several outstanding scientists who became close friends: Ephrussi, Lwoff, Monod, Needham and his wife, J. Runnström, S. Hörstadius and others. At the time, Lwoff and Monod were protozoologists working with E. Chatton on ciiates. Ephrussi was trying to establish whether mitosis is a physical act or a chemical process by meathe respiration of cleaving sea urchin eggs at various temperatures. Runnström, Hörstadius, P.E. Lindahl and younger Swedish workers were experts in sea urchin experimental and chemical embryology. The Needhams, in the company of E. Baldwin and J. Yudkin, were interested in the biochemical mechanisms of evolution. Roscoff was indeed an exciting place for a young medical student eager to become a chemical embryologist. Its busy intellectual atmosphere was a big change from Dalcq's quiet laboratory in Brussels. Returning to my problem of DNA synthesis during cleavage, the biochemical evidence, in contrast to cytochemistry, supported the migration theory. I confirmed old reports that the total nucleic acid content does not increase markedly during sea urchin embryogenesis. On the other hand, both the Feulgen reaction and DNA estimations by the recent biochemical method of Dische demonstrated unambiguously that DNA is synthesized when cells divide. The contradiction between the two sets of data was obvious. Nothing is more exciting for a scientist than to be caught among contradictory facts; nothing is more rewarding for him than to find the right way out of the contradiction. My bold, unorthodox solution was that sea urchin eggs contain large amounts of RNA, a plant nucleic acid; unlike nuclear DNA, this cytoplasmic RNA would not undergo synthesis during egg cleavage. These ideas were so much against common belief that I. sent them to Joseph Needham; he was so puzzled that he took the matter to his chief, the Nobel Prize winner F.G. Hopkins. Hoppy's advice was full of wisdom: "This chap should not care about what is written in textbooks but make experiments to test his ideas!" Back at Roscoff, I looked for the presence of pentoses in sea urchin eggs. My French friends laughed at me for using a method designed for estimation of pentosans in straw, but it turned out that sea urchin eggs indeed contain large amounts of pentoses in the form of an RNA. My teacher Dalcq was not happy with my results; he told me bluntly that he would never believe my RNA story unless I showed him its localization under the microscope. I had great respect for Dalcq, who was an out-standing embryologist and a first-class microscopist. I quickly realized he was right: I would never convince myself of the presence of RNA in animal cells unless I could demonstrate its intracellular localization in tissue sections. After unsuccessful attempts, I worked out, around 1939, a simple method for the cytochemical detection of RNA, based on staining sections with a mixture of two dyes (methyl green and pyronine), the staining's specificity being insured by prior enzymatic digestion of RNA with ribonuclease. This allowed me to convince both morphologists and biochemists that RNA is present in all cells, even bacteria. It is localized in the cytoplasm and the nucleoli—a conclusion drawn independently by T. Caspersson, who had built in Stockholm a very delicate and sophisticated U.V.-cytophotometer. We both concluded that there is a close correlation between the RNA content of a cell and its ability to synthesize proteins. A role for RNA in protein synthesis completely violated the then-prevalent theory that proteins are synthesized by the reversal of proteolysis. When, shortly before the war, Albert Claude isolated small RNA-containing granules, I immediately thought these "microsomes" were the agents of protein synthesis and, with R. Jeener and H. Chantrenne, I tried to prove it. All we could do under wartime conditions was to show that microsomes always contain RNA and are ubiquitous; we amassed circumstantial evidence for a role in protein synthesis that was definitely proven only 15 years later. Sea urchin eggs, by a strange accident, had brought me to the very heart of what later became molecular biology. Our understanding of DNA replication, transcription and translation remained dim, however, until biochemists, biophysicists and geneticists working on simpler systems (bacteria, phages, macromolecules) elucidated their mechanisms. Brachet studied medicine at the University of Brussels and became professor of cytology and embryology at its Faculty of Sciences in 1942. He has been an emeritus professor since 1977.

Rewriting the Book on Nucleic Acids

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