Chargaff's Rules: the Work of Erwin Chargaff

The Separation and Quantitative Estimation of Purines and Pyrimidines inMinute Amounts (Vischer, E. and Chargaff, E. (1948) J. Biol. Chem.176, 703-714) Erwin Chargaff (1905-2002) was born in Czernowitz, which at that time was aprovincial capital of the Austrian monarchy. He graduated from high school atthe Maximilian Gymnasium in Vienna and went to the University of Vienna in1923. “I was eighteen and the world was before me,” notedChargaff. “The future scientist should at this moment be able to tellstories out of his brief past, how he always knew that he wanted to be achemist or a lepidopterist; how he could be nothing else, having blown himselfup at six years of age in his basement laboratory or having captured, intender years, a butterfly of such splendor and rarity as to make Mr. Nabokovblanch with envy. I can offer nothing of the sort. Being gifted for manythings, I was gifted for nothing... It was quite clear to everybody that Ishould have to enter the university and acquire a doctor's degree. This hadthe advantage of postponing the unpleasant decision about my future by fouryears or so and also of equipping me with the indispensable prefix withoutwhich a middle class Austrian of my generation would have felt naked”(1Chargaff E. A fever of reason the early way..Annu. Rev. Biochem. 1975; 44: 1-18Crossref PubMed Scopus (7) Google Scholar). At the university, Chargaff decided to study chemistry. Although he hadnever taken the subject before, it offered the most hope of employment aftergraduation, specifically the opportunity to work at his uncle's alcoholrefinery. Unfortunately, before he even started on his dissertation, the unclewas dead and Chargaff's alcoholic hopes had evaporated. Nonetheless, he stuckwith chemistry and received his doctoral degree in 1928. His dissertation,done under the supervision of Fritz Feigl, dealt with organic silver complexesand with the action of iodine on azides. Because there were very few researchpositions in Austria, Chargaff left for the United States in 1928 as a MiltonCampbell Research Fellow at Yale University. Chargaff recalled, “As thetime of my departure grew nearer, so grew my fears. I was afraid of going to acountry that was younger than most of Vienna's toilets”(1Chargaff E. A fever of reason the early way..Annu. Rev. Biochem. 1975; 44: 1-18Crossref PubMed Scopus (7) Google Scholar). However, he found Americaagreeable enough that he remained there for two years, working with R. J.Anderson on tubercle bacilli and other acid-fast microorganisms. In the summer of 1930, Chargaff returned to Europe and was appointedAssistent at the Bacteriology Department of the University of Berlin.His work in Berlin covered a variety of topics including a study of the lipidsof the bacillus Calmette-Guérin and a detailed investigation of the fatand phosphatide fractions of diphtheria bacteria. However, with the rise ofHitler, Chargaff felt the need to leave Germany, and in 1933 he transferred tothe Pasteur Institute in Paris. During his brief time in Paris, he worked onbacterial pigments and polysaccharides. Then, in 1935 he returned to theUnited States to become an assistant professor of biochemistry at ColumbiaUniversity. Seventeen years later he became a full professor and later waschairman of the department from 1970 to 1974, when he retired to emeritusstatus. In 1944, Chargaff read Oswald Avery's report that the hereditary units, thegenes, were composed of DNA(2Avery O.T. Macleod C.M. Mccarty M. Studies on the chemical nature of the substance inducing transformation ofpneumococcal types. Induction of transformation by a desoxyribonucleic acidfraction isolated from pneumococcus Type 111..J. Exp.Med. 1944; 79: 137-158Crossref PubMed Scopus (1795) Google Scholar). This had a profound impacton Chargaff, as he recollected, “Avery gave us the first text of a newlanguage, or rather he showed us where to look for it. I resolved to searchfor this text. Consequently, I decided to relinquish all that we had beenworking on or to bring it to a quick conclusion”(3Chargaff E. Preface to a grammar of biology..Science. 1971; 172: 637-642Crossref PubMed Scopus (46) Google Scholar). Thus started Chargaff'swork on the chemistry of nucleic acids. He began with the belief that if DNA from different species exhibiteddifferent biological activities, there should also be chemically demonstrabledifferences between the DNA. His immediate challenge was to devise a method toanalyze the nitrogenous components and sugars of DNA from different species.Because large amounts of DNA would be hard to come by, his methods also had tobe applicable to small amounts of material. The formulation of this proceduretook two years and was aided by several recent technological developmentsincluding the introduction of paper chromatography to separate and identifyminute quantities of organic substances and the photoelectric ultravioletspectrophotometer. The paper describing Chargaff's analytical method is reprinted here as aJournal of Biological Chemistry (JBC) Classic. His procedureconsisted of three steps. The first was the separation of the DNA mixture intoindividual components by paper chromatography. Next, the separated compoundswere converted into mercury salts. And finally, the purines and pyrimidineswere identified via their ultraviolet absorption spectra. Chargaff tested themethod on several mixtures of purines and pyrimidines and reported hisencouraging results in the Classic. In a separate paper, printed back-to-backwith the Classic, he put his method to use and analyzed the DNA composition ofyeast and pancreatic cells(4Vischer E. Chargaff E. The compositionof the pentose nucleic acids of yeast and pancreas..J. Biol.Chem. 1948; 176: 715-734Abstract Full Text PDF PubMed Google Scholar). A month later, Chargaff submitted two additional papers to the JBC on thecomplete qualitative analysis of several DNA preparations. The first paperdealt with the purines and pyrimidines of the DNA of calf thymus and beefspleen (5Chargaff E. Vischer E. Doniger R. Green C. Misani F. The composition of the desoxypentose nucleic acids ofthymus and spleen..J. Biol. Chem. 1949; 177: 405-416Abstract Full Text PDF PubMed Google Scholar) and the second withthe DNA of tubercle bacilli and yeast(6Vischer E. Zamenhof S. Chargaff E. Microbial nucleic acids: the desoxypentose nucleic acids of avian tuberclebacilli and yeast..J. Biol. Chem. 1949; 177: 429-438Abstract Full Text PDF PubMed Google Scholar). Although these paperswould eventually prove to be invaluable contributions to our understanding ofthe structure of DNA and the genetic code, they were almost not published.“One curious circumstance attending the publication of these papersdeserves mention because it illustrates the ignorance about nucleic acids thatthen prevailed among the scientific elite,” wrote Chargaff. “Ihad, at that time, already published something like 75 articles in theJournal of Biological Chemistry without ever having one sent back bythe editor for clarification or revision. The papers about DNA composition,however, were returned to me with a particularly silly objection. How could I,the editor asked, express the composition of a DNA as moles of adenine orguanine, cytosine or thymine, per gram-atom of phosphorus, since the purinesand pyrimidines did not contain any phosphorus? After I had repeated, in myanswer to the editor, part of the introductory lecture on the nucleic acids,which at that time I was already giving to the first-year medical students atColumbia, we achieved grudging reconciliation”(7Chargaff E. How genetics got a chemicaleducation..Ann. N. Y. Acad. Sci. 1979; 325: 345-360Crossref Scopus (37) Google Scholar). Over time, Chargaff improved on his initial quantification methods byintroducing formic acid hydrolysis for the simultaneous liberation of allnitrogenous constituents and by using a UV lamp to demonstrate the separatedadsorption zones on the filter strip. These improvements permitted him torapidly analyze DNA from a variety of species. Eventually, Chargaff summarizedhis findings on the chemistry of nucleic acids in a review in 1950(8Chargaff E. Chemical specificity of nucleicacids and mechanism of their enzymatic degradation..Experientia. 1950; 6: 201-209Crossref PubMed Scopus (201) Google Scholar). His two main discoveries,(i) that in any double-stranded DNA the number of guanine units equals thenumber of cytosine units and the number of adenine units equals the number ofthymine units and (ii) that the composition of DNA varies from one species toanother, are now known as Chargaff's Rules. These results provided the firmevidence needed to disprove the prevailing tetranucleotide hypothesis. Thehypothesis, originally put forth by JBC Classic author Phoebus Levene(9Classic JBC Levene P.A. J. Biol.Chem. 1919; 40 (http://www.jbc.org/cgi/content/full/277/22/e11): 415-424Google Scholar), stated that DNA wascomposed of a large number of repeats of a GACT tetramer, which was obviouslyno longer valid. Chargaff's research also helped lay the groundwork for JamesWatson and Francis Crick's discovery of the double-helix structure ofDNA. 1All biographical information on Erwin Chargaff was taken from Refs.1Chargaff E. A fever of reason the early way..Annu. Rev. Biochem. 1975; 44: 1-18Crossref PubMed Scopus (7) Google Scholar,3Chargaff E. Preface to a grammar of biology..Science. 1971; 172: 637-642Crossref PubMed Scopus (46) Google Scholar, and7Chargaff E. How genetics got a chemicaleducation..Ann. N. Y. Acad. Sci. 1979; 325: 345-360Crossref Scopus (37) Google Scholar. 1All biographical information on Erwin Chargaff was taken from Refs.1Chargaff E. A fever of reason the early way..Annu. Rev. Biochem. 1975; 44: 1-18Crossref PubMed Scopus (7) Google Scholar,3Chargaff E. Preface to a grammar of biology..Science. 1971; 172: 637-642Crossref PubMed Scopus (46) Google Scholar, and7Chargaff E. How genetics got a chemicaleducation..Ann. N. Y. Acad. Sci. 1979; 325: 345-360Crossref Scopus (37) Google Scholar.