Reminiscences about nucleic acid cytochemistry and biochemistry

There was sharp opposition between ‘analomists and ‘physiologists’ when I was a medical student in the University of Urussels, some 60 years ago. This split was cxcmplificd by the prcsencc of two separate buildings. called respcctivcly Institutes of Anatomy and of Physiology, in the newly crcctcd Medical School. The first housed embryologists, histologists and pathologists, the second physiologists, biochemists, bacteriologists and pharmacologists. Uiochcmistry was a rcccnt outgrowth of the older and larger physiology laboratory; the young professor. E. J. Bigwood, was at that tirnc mainly interested in redox potcntials. Thcrc was no inner communication bctwccn the two b11ildings except a long dark underground corridor; wc called it the ‘tunnel’. Students used it, but in gencral senior ‘anatomists” and ‘physiologists’ wcrc not much intcrcstcd in meeting each other. I became an ‘anatomist in 1927, although I had a much greater interest in organic chemistry than in human bones. We had been told by our professor of histology, I’ol Gerard, that in merotomy cxpcri1ncnts (bisection of an egg or unicellular organism) anuclcatc cytoplasmic fragments survive and even display normal activities for some time. This fascinated mc and 1 dccidcd to study the interactions between nucleus and cytoplasm in intact cells (I a1n still working on them today). This choice led me to the embryology laboratory headed by my father, who very wisely advised mc to work under his young colleague, Albert Dalcq. Dalcq had been among the very first to dcmonstratc that calcium ions are of paramount importance for the maturation and fertilization of starfish eggs; he was then analysing the respective roles of the sperm and egg nuclei in frog development by X-irradiation and local treatment with trypaflavine. I-fis expcrimcnts showed that non-nucleated fertilized eggs can undergo a few irregular clcavages, but never gastrulation. I was lucky to work with Dalcy bccausc, in those days, he displayed a real intcrcst in biochemistry. He had even spent a couple of months in David Kcilin’s Ii1boratory in Cambridge whcl-e he had Icarncd a few biochc11iical tccliniqucs, with the hope of following cytochronic synthesis during devclopmcnt. Hut hc soon rcalizcd that hc was and would always rcniain a morphologist; hc was fond of cytochemistry, enjoying his microscopic investigations of the localization of phosphatases in egg and sperm; hc would ncvcr have crushed an egg for the analysis of biochemical paramctcrs (cvcn for phosphatasc ilctiVi\y iiicasiircmcnts). As soon as I had Icarncd the classical histological techniques of fixation, cmbctlding, sectioning and staining, Dalcrl proposed a research subject for mc: a study of the localization of ‘thymonuclcic acid’ in growing oocytcs with the rcccnt cytochcmical method of Fculgcn and Rosscnbeckl. According to the biochemistry textbooks, then as now, there are two main classes of nucleic acids: one of them. 11ow known as DNA, had a C~ICCI sugar residue which was identified only in 1930 as deoxyribose by Levenc. Mikcska and Mori*. This category of nucleic acids was believed to bc localized in the nuclei of only animal cells; tlic prototypc of these ‘animal nucleic acids’ was thymonuclcic acid from calf thymus. The other type of nucleic acid (our RNA). known to contain a pcntose residue that was Iatcr identified as o-ribosc, was thought to bc specific to plant cells. Yeast zymonucleic acid was the bcstknown of these ‘plant nucleic acids’ (also called phytonucleic acids). The role played by the two kinds of nucleic acids in tlic nuclei was mysterious: their small size (they were bclicvcd to 1~)~ tctranuclcotidcs of about I.300 Da) l~rccIutlcd any gcnctic function; it was suggcslcd that they might act as intracellular buffcrs3 01 as colloids giving a high viscosity to the nuclciJ. This was all I could find about nucleic acids in biochemistry textbooks around 19.30. R. Fculgcn was a distinguished biochemist who had tried for many years to identify the mysterious sugar present in thymonucleic acid (DNA): he discovered that this sugar gives aldehyde reactions and thought that it was glucal, an aldchyde derivative of glucose. Fculgcn also found that DNA rcactswith fuchsin sulfurous acid (the classical Schiff aldchydc reaction) to give a violet compound after removal of the purincs by mild acid hydrolysis. Finally, hc applied this aldehydc colour reaction to tissue sections after fixation of the cells with a rather harsh fixative (a mixture of saturated sublimate and acetic acid). Feulgen’s main important result was that Nil Cell nuclei, Vegctal as Well as alliIni1l, stained positively with his procedure. l-lowcvcr, this very important finding (DNA is present in all cell nuclei) was not taken seriously by many biochemists who bclicvcd in colour reactions obtained in test tubes, but not on tissue sections. Their scepticism incrcascd when Fculgcn showed that, under ccrtain conditions, the cytoplas1n also gave a Schiff reaction due to a class of lipids, the plasmalogcns. He made a sharp distinction (which remains true today) bctwcen the ‘nucleal’ reaction for DNA and the ‘plasmal reaction for plasmalo-