An Interview with Manuel Varela and Ann Varela—Erwin Chargaff—Precursor to Watson and Crick?

Oct 17, 2020 by

Michael F. Shaughnessy

  1. Erwin Chargaff had a kind of tumultuous early childhood—Where was he born, and what happened?

Prof. Erwin Chargaff is a pioneer molecular biologist who established the nucleotide base binding properties for DNA, namely, the A—T and G—C associations. In 1905, Chargaff was born on August 11 in Czernowitz, Duchy of Bukovina, Austria-Hungary, now Ukraine. Chargaff’s father, Hermann, was a wealthy bank owner until some employees embezzled the funds. Both parents were well educated. His mother’s name was Rosa Silberstein. Chargaff had a younger sister named Greta.

Around the age of nine, Chargaff’s family was vacationing at the Baltic Sea resort during World War I. The hope of returning home was dashed because the Russian Army was invading and about to seize their hometown; thus, Chargaff’s family moved to Vienna.

Now that the family lived in Vienna, he was fortunate enough to attend the Maximiliansgymnasium (now Gymnasium Wasagasse), one of Vienna’s finest schools of culture. There he learned both Greek and Latin, which was impressive considering he was evidently a late talker. In toll, he astoundingly became proficient at fifteen languages.

Chargaff’s studies at the Maximiliansgymnasium focused on natural history and the arts. Listening to operas and discussing poetry and literature were common pastimes with his friend, Albert Fuchs. It was up to him to supplement his scientific knowledge. He was an avid reader of Western classical literature, and he was a member of the Boy Scouts.

Chargaff’s father died in 1934, and his mother’s fate is unknown, as the Nazi’s took control of Austria in the late 1930s. Chargaff was unsuccessful in 1943 at bringing his mother to the United States, which, understandably, made him melancholy for the rest of his life. Chargaff later wrote that she died, “only God knows where and when having been deported into the nothingness from Vienna in 1943.”

2) Chargaff’s Law—what exactly is it, and why is it important?

Some sources regard Chargaff’s Law as the phenomenon of complementary paring for the nucleotide bases that constitute double-stranded DNA. The Law has its origins in the four rules or observations that Chargaff published in the late 1940s and early 1950s. Chargaff’s Law is identical to one of these four rules. These four observations are discussed below in your next inquiry.

The Law deals with the nature of the interactions between the nitrogenous bases of duplex DNA. This base pair rule established by Chargaff states that in DNA of most, if not all, living organisms on Earth, the purine bases like to form specific pairs with the pyrimidine bases. The pyrimidine bases include cytosine and thymine, and purine bases include adenosine and guanine. The Law further specifies that cytosine complements guanine, and hence they form a base pair. Likewise, thymine will always create a base pair with adenosine.

The Law of Complementary base pairing can be astutely used in molecular biology. For example, suppose one knows the nitrogenous bases’ sequence along the length of a single-stranded DNA molecule. In that case, one immediately knows the corresponding base sequence of the complementary strand of the DNA.

Such base sequence knowledge can come in handy if one can access one DNA strand’s base sequence. Investigators can deduce the base sequence of the second strand of DNA by examining the sequence of bases in just one of the DNA strands.

Secondly, by exploiting Chargaff’s Law, an investigator may determine whether one deals with a single or a double-stranded DNA molecule in a sample of DNA of unknown origin. Such information would be necessary if, for example, an investigator discovers a virus. The nucleic acid genomes of some viruses are single-stranded while others are double-stranded. Thus, the study of base-pairing laws can help the investigator understand whether an unknown virus’s genome is single- or double-stranded from a so-called complementary perspective. If the new virus is single-stranded, it limits the number of viral families to which the unknown virus belongs. Likewise, suppose an investigator finds that an unknown virus’s genome is double-stranded. In that case, the investigators know to consider those viral families in which their genomes are double-stranded.

3) Chargaff found that amounts of a) guanine, b) cytosine, c) adenine, and d) thymine seem to vary by species. Why is this important—and what do each of these contribute to human functioning?

Dr. Chargaff would become famously known for his four rules or conclusions. These rules are based on his experimental work dealing with the DNA base composition analyses for samples taken from many different organisms.

Regarding his experiments, Chargaff extracted cells from an astounding array of living organisms. He focused on as many species of organisms as he could get his hands on. The living forms ranged tremendously in their diversity, from bacteria to fungi, amphibians, fish, birds, and even cells from the human liver. For each type of the living cells collected, Chargaff extracted the chromosomal matter contained therein and placed the cellular genomic materials in liquid solutions. Next, Chargaff removed the protein that was stuck to the chromosomal DNA samples for each cell type.

Focusing on the remaining nucleic acid material that remained in the chromosome preparations, Chargaff added a strong acid. The highly acidic solution hydrolyzed the DNA and released the nitrogenous bases off of the DNA backbone. The acidification process left the individual nucleotides intact, which permitted the performance of the DNA base composition analyses.

To learn the chromosomal base composition for each of the organisms’ cells, Chargaff then needed to separate the released bases from each other. The nucleotide separation process had to be performed individually for every cell type that he found. To separate the nucleotide molecules, Chargaff used a method called paper chromatography.

To conduct paper chromatography, Chargaff applied his base samples onto a paper-based matrix material. The bases then migrate through the matrix structure of the paper. The DNA bases moved through the paper matrix with special migration rates for each nucleotide, depending on the degree of interaction between the base type and the matrix.

Whether A, G, C, or T, each base type produced distinctive banding patterns on the chromatographic paper. Typically, the A base moved the furthest, followed by C, then G, and T driving the least distance from the chromatography paper’s point of origin. Chargaff used scissors or a razor blade to cut out individual bands from the chromatograph and added to test tubes to produce unique nucleotide solutions, one chemical solution for each of the four nucleotide bases.

File:Chromatography tank.svg

https://commons.wikimedia.org/wiki/File:Chromatography_tank.svg

Figure 26. Diagram of a thin layer chromatography apparatus.

The paper chromatographic apparatus, seen in Figure 26, consisting of a (1) lid for coverage, (2) the chromatography paper, and a tank (4) to hold the solvent. As the solvent migrated through the chromatographic paper, it formed a so-called solvent front, (3), taking the DNA bases along with it. The DNA bases would migrate along the paper matrix’s length, forming visible “bands” on the chromatograph.

Chargaff then used a piece of lab equipment called a spectrophotometer to measure the amounts of each DNA nucleotide base. Each base type from DNA absorbed light at a given wavelength, and from each of these nucleotide light-absorption profiles, Chargaff could calculate the amounts of each DNA nucleotides. Lastly, Chargaff compared the nucleotide base compositions for every living specimen he collected. From these DNA base composition data, Chargaff made four insightful observations, his rules.

The first rule was that the nucleotide base compositions of DNA were different from one species to the next. The second observation made by Chargaff was that DNA base composition was identical in various tissues and organs of the same species. The third rule of Chargaff was that the DNA nucleotide composition in a particular species remained the same regardless of the age, nutritional status, or environment.

As mentioned above, one of these four rules would later become known as Chargaff’s Law. The fourth observation of Chargaff would become his Law, for which he would become forever associated.

Chargaff’s rule number four (his Law) stated that regardless of the species, all cellular DNA contained equal amounts of A and T and equal amounts of G and C. When Chargaff analyzed the fourth rule’s implications, he astutely deduced that the sum of purines (G and A) equaled the sum of the pyrimidines (T and C). Therefore, G + A = T + C.

4) To have a “law” named after one is surely a major accomplishment. How does this “law” impact us today? Is it still referred to today?

Chargaff’s Law is still relevant in modern times. Sometimes called Chargaff ratios or the equivalence rule, it is virtually common knowledge today that T forms a base pair with A and that C base pairs with T. This Law forms the root basis of molecular biology and modern genome projects. Because we know the sequence of one strand of DNA for a gene, we automatically see the complement, thanks to Chargaff and his Law. Furthermore, we learn the messenger RNA code, which allows one to see the protein sequence of amino acids on a peptide, thanks indirectly to Chargaff.

The base-pairing properties between A—T and G—C are universal for most living organisms on Earth. Because of the universality inherent in the base-pairing rules, we can decode the entire genomes for organisms using new bioinformatics tools, studying coding sequences and molecular structures using high throughput electronic computation. Chargaff’s Law of nucleotide base-pair complementarity permitted all of these advances to come to fruition. See Figure 27.

File:DNA Structure+Key+Labelled.pn NoBB.png

https://commons.wikimedia.org/wiki/File:DNA_Structure%2BKey%2BLabelled.pn_NoBB.png

Figure 27. The DNA structure shows the four bases, adenine, cytosine, guanine, and thymine.

With time and with each new advancement in molecular-based technology, it is increasingly possible to have one’s genome mapped completely for the locations of one’s entire gene composition. It is also increasingly likely to have the complete nucleotide sequence determined for a genome of an individual. Further, one can use the base-pairing laws to examine the phylogenetic relationship and the individual’s genetic history. With genealogical projects on the rise, DNA figures prominently in the popularity of these endeavors.

From a forensic science point of view, the remains of a corpse or a crime victim can be identified by routinely invoking the rules of Chargaff. Perhaps even the perpetrator of a crime can be discerned with the molecular tools, all made available because of Chargaff and his four principles. An unknown species can be known. Suppose one finds a fossil or a decomposed body, the specimen’s identity can be made known, as well. A new paternal relationship can be established, such as when a child’s parent is unknown. Such relationships can be brought out into the light.

5) Apparently, Chargaff met Watson and Crick—any details as to that meeting?

There are different accounts of this now-famous meeting between Erwin Chargaff himself and Jim Watson and Francis Crick. You will recall that Watson and Crick would become world-famous for proposing a novel and accurate model for the three-dimensional structure of DNA. However, before their fame, they were neophytes who were grappling with the seemingly phenomenological data from various sources, Chargaff’s data among these. The three investigators would agree that Chargaff’s carefully collected and analyzed data would definitively lead Watson and Crick to conclude that A binds to T and C binds to G. Chargaff’s data implied as much, and Crick and Watson ran with it. Based on the relationships established by Chargaff for the interactions between A—T and G—C, Crick and Watson reckoned that these base-pair relationships were true and would thus fit nicely in the middle of DNA. If so, then one last deduction was required in which the sugar-phosphate strands ran in opposite directions, i.e., in an antiparallel fashion. Once the bond angle calculations supported the structural model’s energetics, the rest of the structural components fell into place.

The first meeting between Chargaff and the Crick-Watson duo has been considered by all three scientists in their autobiographical writings. Crick wrote that when shown a draft of their second Nature manuscript, which dealt on implications of the DNA model, Chargaff was reported to have replied that while their first paper on the DNA structure was “interesting,” their second paper “was no good at all.” According to Crick, Chargaff would tell the same opinion to others, such as the famous Fritz Lipmann, who would be convinced only after listening to Crick himself during one of his lectures. In a film on the topic produced by the BBC, Crick was a consultant. The film covered the basics of the so-called disastrous conversation between the Watson-Crick team and Chargaff. The dinner scene’s scientific details were not contested in Crick’s memoir, “What Mad Pursuit.” Crick would only write that their infamous meeting with Chargaff was not a college dinner. Further, Crick would also claim that Chargaff’s influence with his rules was not at the forefront of Watson’s thinking when he stumbled upon the correct base-pairings between A and T and between G and C.

In retrospect, Chargaff’s influence in leading Watson and Crick to the correct DNA structure is undeniable. Watson would later write that he did indeed consider Chargaff’s base composition data in his classic, “The Double Helix.” In fact, in his second memoir, “Avoid Boring People: Lessons from a Life in Science,” Watson chastised Linus Pauling for, among other things, not considering Chargaff’s data regarding DNA. Linus Pauling confirmed the sentiment. Pauling’s version of the DNA story would lament not incorporating Chargaff’s base ratio data into his proposed structure.

Watson’s account of their famous meeting with Chargaff was recorded in “The Double Helix.” Crick and Watson were invited by John Kendrew to join him and Chargaff for drinks after dinner that evening at the Peterhouse in Cambridge. In Kendrew’s room, the conversation started amicably, but, according to Watson, ended in disaster for the Crick-Watson team. The disastrous turn took hold when Kendrew nonchalantly mentioned that Watson and Crick were solving the DNA structure problem using molecular models.

According to Watson’s account, Chargaff was not amused—accusing them of being “dark horses” trying to win the race to the double helix. Kendrew attempted to control the damage by remarking that Watson was not your “typical American,” which, according to Watson, merely served to produce more scorn from Chargaff.

According to Watson, Chargaff derided his accent and long hair. Watson replied that he kept his hair as such to avoid confusion with the American military servicemen, a statement which, according to Watson, merely proved his “mental instability” in Chargaff’s mind.

The well-known meeting with Chargaff then took a turn for the worse, when Watson and Crick could not recall the correct chemical structures on paper for the various nucleotides. The meeting ended unceremoniously. Watson, Crick, and their followers would forever write about Chargaff in unflattering terms.

In 1978, Chargaff would report his version of the legendary encounter with the “dark horses” in his autobiographical memoir titled “Heraclitean Fire: Sketches from a Life Before Nature.” Chargaff stated they met after lunch, not dinner, as Watson reported. Chargaff also related that a lack of basic chemistry failed to stymie Crick and Watson in their efforts to fit DNA into a double helical structure. He thought they had been influenced by Linus Pauling’s discovery of the alpha-helix structure found in proteins. During the meeting, Chargaff confirmed adenine in close association with thymine and guanine with cytosine. The base-pairings formed arrangements that could fit within a double helix’s confines.

Chargaff later stated that his base ratio data directed Watson and Crick to the correct structure. Chargaff further reported that if Watson and Crick had any notions about base-pairing rules as implicated by his findings, they had concealed any knowledge of it during their encounter. Not surprisingly, Chargaff continued, Watson and Crick seemed not to know much of anything. In an interview of Chargaff by science historian Horace Freeland Judson, Chargaff would state that never had he met two individuals (Crick and Watson) who knew so little but aspired so much and that they worked so “little” but talked “so much” and in Crick’s case, a lot of “nonsense.” Chargaff recalled hearing so much about “pitches” (referring to structural dimensions of the double helix) that he spoke of Watson and Crick as “pitchmen in search of a helix.”

6) Often, scientific edifices are built on the solid foundations of others. Would you say this is true of Chargaff? 

In a word, yes. Not only did Chargaff’s work serve as a firm foundation to the elucidation of the correct DNA structure, but Chargaff’s influential base-pairing studies were also helped by the investigations of others before him. For example, in 1944, Archer J.P. Martin and Richard L.M. Synge developed the paper chromatography method that Chargaff would put to good use in determining his base ratios data. Furthermore, Roger Vendrely and André Boivin discovered that the nuclei of any given individual’s cells contained the same DNA concentrations. Vendrely and Boivin further found that in sex cells, eggs and sperm, the DNA amounts were approximately half of those found in somatic cells. These DNA content consistency findings in the cell and germ cell nucleus harboring half the somatic cell content became known as the Vendrely—Boivin rule. Chargaff would perceptively incorporate these foundational studies into his influential work.

Likewise, the four influential observations of Chargaff would most certainly be instrumental in the proposal of the correct DNA structure by Watson and Crick. The base-pairing rules are still used in modern times on many fronts and levels. Chargaff’s observation about the base composition of DNA and their relationship to specific species effectively determined the species using DNA before the advent of sequencing technologies. The universality of an individual’s cellular DNA content was tremendously influential in aiding investigators to map the genome of many bacteria, plants, animals, and humans. Chargaff discovered that nucleotides’ base composition in organisms’ DNA did not change with the environment, metabolism, or age. The proclamation proved to be persuasive towards the development of modern genetics, especially that of humans. The experimental studies of Chargaff will undoubtedly continue to be powerful in its scientific foundations for the next millennia.

One interesting story features Chargaff and Maurice Wilkins. Chargaff had provided a sample of DNA to Wilkins. Nevertheless, to his dismay, Wilkins found that the DNA was not amenable to crystallization. The DNA work of Wilkins, therefore, had gone nowhere during the early years of the 1950s. On the other hand, the DNA sample provided to Rosalind Franklin by Rudolf Signer from Bern had been perfect for X-ray crystallographic studies.

7) In later years, Chargaff seemed to believe that genetic engineering would have extremely difficult, problematic consequences. What was he thinking in this regard?

Indeed, Chargaff was to become a vocal opponent of genetic engineering. He warned about the potential dangers of genetic engineering technology. He had spoken of potentially dire consequences of creating new artificial forms of life in the laboratory using genetic engineering tools. Chargaff began a written campaign about his views on genetic engineering. He noted that though one might stop splitting the atom or decide not to kill entire human populations; one would be unable to recall a new genetically engineered life form. The new genetic chimeras, Chargaff continued, would survive our children, their children, and us. The microbes used in genetic engineering, such as the SV40 virus, were natural cancer agents in laboratory animals. These sorts of proclamations and others published in the mid-1970s by Chargaff in the journal Science raised the ire of many prominent molecular biologists of the day.

The pushback was fierce. Chargaff’s views were described as “absurd” by notables such as Paul Berg (chapter 4), whose work featured prominently in the development of recombinant DNA. Cancer biologist and virologist Renato Dulbecco, featured in our 2020 book “An Overview of Biomedical Scientists and their Discoveries,” was so frustrated that he offered to drink a flask of SV40 to show that it did not cause cancer in humans.

8) While never winning the Nobel Prize—he did author many books—some in English and some in German. What were his contributions there?

As mentioned above, in 1978, Chargaff penned a candid autobiography titled “Heraclitean Fire: Sketches from a Life Before Nature.” The title has its origins in Heraclitus of Ephesus (b. 540 B.C.), who wrote about a “universal flux” of fire as the primary material and had a contemptuous tone. Chargaff would adopt this tone in his dealings with others.

Some historians note that perhaps because Chargaff had been enormously disappointed in being passed over for the Nobel, especially when Watson, Wilkins, and Crick took the honor in 1962 that Chargaff forever turned against genetic engineering. Chargaff was miffed about the omission by the Nobel commission. He had felt that without his DNA base ratio data, Watson and Crick would have been hard-pressed to fit the A—T and G—C base pairs into the dimensions of a DNA double-helical structure model.

Chargaff also criticized the ethical practices of Max Perutz in providing John Randall’s DNA report to Watson and Crick. Although overlooked by the Nobel Committee, Chargaff’s contributions were extensively recognized elsewhere. Chargaff was nominated and elected to the National Academy of Sciences in 1965. He received many awards, including the Pasteur Medal in 1949, Carl Neuberg Medal in 1958, Charles Leopold Mayer Prize in 1963, Heineken Prize in 1964, Gregor Mendel Medal in 1974, and the National Medal of Science in 1975. Chargaff’s scientific articles and books are extensive. A brief list of his papers can be found at the end of this chapter.

9) Have I neglected to ask anything about this scientist, who fortunately escaped Hitler and the Holocaust?

Chargaff attended Vienna College of Technology and studied at the University of Vienna from 1924–1928, to defend his Ph.D. in chemistry. The focus of his thesis was about organic silver complexes and the reaction of iodine with azides. His advisors and professors did not seem very enthused with Chargaff’s interest in reading publications such as the American Chemical Society Journal. They said, “There wasn’t anything useful in it.” Studying abroad in America was also discouraged. Naturally, applying to the Milton Campbell Research Fellowship in Organic Chemistry in America was foremost on his mind.

Chargaff’s entrance in the U.S. was fraught with misunderstanding by the immigration officials in New York. They did not comprehend how someone whose passport described him as a Ph.D. would be coming to work on a “student visa.” Therefore, they dispatched him to Ellis Island for a few days until he was released into the custody of Treat Johnson, the Yale chemistry department head at Yale University.

From 1925–1930, Chargaff served as the Milton Campbell Research Fellow in organic chemistry. He studied the chemical configuration of the avian tubercle bacilli, Mycobacterium avium, along with long-chain fatty acids. He and his colleague Rudolph Anderson published seven papers.

Chargaff met his future bride, Vera Broido, in 1928 at the Vienna College of Technology. She died in 1995. The couple had a son named Thomas. Unhappy with life in the U.S., Chargaff and his wife returned to Europe in 1930, and he began an assistant position at the University of Berlin. He was to oversee the chemistry department’s research in bacteriology and public health until 1933.

Chargaff’s departure from the University of Berlin was due to Nazi policies against Jews. He then relocated to Paris to continue research at the Pasteur Institute from 1933–1934. In 1935, Chargaff immigrated to the United States and settled in New York. Chargaff and his wife became United States citizens in 1940.

Chargaff accepted a position as a research assistant in the department of biochemistry at Columbia University until 1974. After he retired from Columbia in 1974, Chargaff moved to Roosevelt Hospital, New York, where he stayed until 1992. He died in 2002, on June 20 in Manhattan, New York, at the age of 96.

For additional information about this remarkable scientist and future understanding, visit:

https://academictree.org/chemistry/publications.php?pid=62493

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