Chargaff’s Rules

Erwin Chargaff, 1930.

Erwin Chargaff, 1930.

“We have created a mechanism that makes it practically impossible for a real genius to appear. In my own field the biochemist Fritz Lipmann or the much-maligned Linus Pauling were very talented people. But generally, geniuses everywhere seem to have died out by 1914. Today, most are mediocrities blown up by the winds of the time.”

-Erwin Chargaff, 1985.

Erwin Chargaff, (1905-2002) a biochemist born in Austria, became interested in DNA earlier than most. In the 1930s, while he was working with the bacteria Rickettsi, he became aware of nucleic acids, and decided to educate himself about them.

In 1944, after Oswald Avery published his paper detailing the transforming principle of the Pneumococcus bacteria, Chargaff decided to devote his laboratory almost entirely to the chemistry of nucleic acids. Experimenting with these delicate substances was not an easy task, but eventually a chromatographic technique was developed that would allow for the separation and analysis of the base rings in DNA. This work would later lead to the development of Chargaff’s Rules, the topic of today’s post.

The guanine-cytosine base pair.

The guanine-cytosine base pair.

DNA has two main structural components – a backbone made up of sugar and phosphate groups, and a series of bases found in the middle of the molecule. There are four different bases found in DNA: Adenine (A), Cytosine (C), Guanine (G), and Thymine (T). These four bases can be divided into two categories, pyrimidines and purines. The pyrimidine bases, Cytosine and Thymine, contain only one ring, while the purine bases, Guanine and Adenine, contain two rings. In the DNA structure, the bases pair complementarily, meaning that a purine base will bind with a pyrimidine base. More specifically, Adenine binds with Thymine and Cytosine binds with Guanine.

The adenine-thymine base pair.

The adenine-thymine base pair.

Although this information is now considered fundamental biology, it wasn’t fully understood until after Watson and Crick discovered the structure of DNA in 1953. However, Chargaff’s research in the late 1940s had suggested that the four bases paired in the manner described above.

When Chargaff first decided to devote his laboratory to nucleic acids, he allowed a postdoctoral student named Ernst Vischer to choose his research program from a list of suggested topics. Vischer decided to analyze the purines and pyrimidines in nucleic acids, and went to work developing the chromatographic technique so crucial to isolating the bases. Although his technique was rather crude, it did the trick and Vischer achieved great success. The results of the base analysis showed that the amounts of Adenine and Thymine were about equal, and also that the amounts of Guanine and Cytosine were about equal. Eventually, Chargaff came to the conclusion that in a single molecule of DNA, Guanine/Cytosine = Adenine/Thymine = 1. This concept would later become known as Chargaff’s Rules.

Chargaff’s Rules were officially announced in a lecture delivered in June of 1949 and were first published in May of 1950. However, Linus Pauling had heard about the ratios much earlier – straight from Chargaff in late 1947, while traveling to England for his six-month stay as a professor at Oxford University. Pauling, who considered the trip by ship across the Atlantic Ocean with his family to be a vacation, did not pay attention to what Chargaff told him.

Crellin Pauling, the youngest child of Linus and Ava Helen Pauling, mentioned the remarkable background to the incident in a speech given during a symposium to celebrate Pauling’s life that was held here at Oregon State University in 1995.

Crellin Pauling on “The DNA Story: A Missed Opportunity.”

[Click here to view the rest of Crellin’s talk]

Over time Chargaff mentioned his work to individuals beyond Pauling. In the spring of 1952, Chargaff met James Watson and Francis Crick.  A prickly character, it is clear that Chargaff didn’t think much of the duo. In his truly remarkable autobiography Heraclitean Fire: Sketches from a Life before Nature, Chargaff calls Watson and Crick “a variety act” and further describes them as:

One 35 years old (Crick), with the looks of a fading racing tout. . .an incessant falsetto, with occasional nuggets gleaming in the turbid stream of prattle. The other (Watson), quite undeveloped. . .a grin, more sly than sheepish. . .a gawky young figure.

He further notes that:

I never met two men who knew so little and aspired to so much. They told me they wanted to construct a helix, a polynucleotide to rival Pauling’s helix. They talked so much about ‘pitch’ that I remember I wrote it down afterwards, ‘Two pitchmen in search of a helix.’

[More samples from Chargaff’s acid pen are available here]

Regardless of what he thought of them, Chargaff still mentioned his work to Watson and Crick. The information, although published almost two years earlier, seemed to be new to the pair.

Though Chargaff himself didn’t speculate much on his rules, and Pauling completely ignored them, they did prove to be extremely useful to Watson and Crick. With this new knowledge, the feedback they had received from Rosalind Franklin and Maurice Wilkins, and data obtained through their own research, Watson and Crick were soon able to correctly deduce the structure of DNA.

For more information on DNA, please visit the Race for DNA website, or check out the other posts in the DNA series. For more information on Linus Pauling, visit the Linus Pauling Online portal.

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Oswald Avery’s Pneumococcus Experiments: Forerunner of the DNA Story

Portrait of Oswald T. Avery, ca. 1940s.

Portrait of Oswald T. Avery, ca. 1940s.

DNA, although now known to be extremely important, was overlooked for quite some time. Until early 1953, around when the Watson and Crick structure of DNA was published, most major scientists thought that proteins, rather than DNA, were probably the site of the gene.

In the early 1940s however, experiments performed by Oswald T. Avery and his colleagues at the Rockefeller Institute for Medical Research made a strong argument for DNA as the source of the genetic material. Unfortunately, for many years not much attention was paid to Avery’s work.

Streptococcus pneumoniae, also called pneumococcus, was the subject of Avery’s experiment. This bacterium causes a variety of diseases, including pneumonia and peritonitis. The organism can be found in two forms, smooth (S) and rough (R) which are designated as such simply because of their appearance when viewed microscopically. The smooth appearance is a result of the formation of a polysaccharide capsule that encases the bacterial cell. This capsule protects the cell from immunological defenses, which makes the S form virulent. The R form, on the other hand, is mutated so that it does not synthesize the enzyme that creates the polysaccharide capsule, and is therefore not virulent.

The pneucmococcus bacteria can be further characterized into types, which are designated by roman numerals. Although an S form bacterial cell can be experimentally changed to an R form (and vice versa) provided the cell is not too far degraded, change of type never suddenly occurs. For example, a type III S cell can be converted to a type III R cell, but a type II cell will never spontaneously convert to a type III cell.

Although a spontaneous change of type is not possible, a specific experiment had been done showing that a transformation of type can be induced. This experiment was first performed by injecting a live culture of the Type II R form into mice along with a dead culture of the Type III S form. Theoretically, none of the mice should have died because they hadn’t been exposed to a virulent form of the bacteria. However, many of the mice did actually die, and living Type III S form bacteria was extracted from their blood. Later, the same experiment was accomplished by growing the bacteria in a glass dish rather than in mice.

Understandably, this transformation from a non-virulent bacteria to a virulent bacteria was troubling to the medical community. Although some scientists may have been concerned with the mechanism of transformation, Oswald Avery was more concerned with the identity of the agent performing the transformation. He went to work on devising an experiment that would allow him to isolate the transforming agent from the rest of the bacterial cell. Although DNA extraction is now considered a simple process, it was just beginning to emerge during the time when Avery began his work. The fact that Avery did not know that the transforming agent was in fact DNA complicated matters even further.

Nevertheless, after years of hard work, Avery and his colleagues were able to develop an experiment that effectively isolated the transforming agent from the bacterial cells. Type III S form bacteria were grown in large vats of broth made from beef hearts. The bacteria was then killed, and washed with brine in order to remove the polysaccharide capsule and whatever protein would come off in the process. The remainder of the bacteria was then precipitated in pure grain alcohol. After this, the precipitate was washed with chloroform and subjected to a digestive enzyme, both of which functioned to remove the remaining protein. Finally, after no trace of protein was evident, pure grain alcohol was once again added, which allowed the transforming agent to be separated. The process was long and difficult, and in the end only yielded approximately ten to twenty-five milligrams of the agent per seventy-five liters of culture.

Transformation of pneumococcal types, from Avery's 1944 paper.

Transformation of pneumococcal types, from Avery's 1944 paper.

After obtaining enough of the active transforming agent to conduct his tests, Avery and his colleagues set out to show exactly what the substance was. First, standard qualitative tests for proteins were performed, which came back negative. Qualitative tests for DNA, however, were strongly positive. Chemical analysis of the substance also showed that the ratio of nitrogen atoms to phosphorous atoms was approximately 1.67 to 1. This number is very close to the DNA ratio, and would have been different had there been a significant amount of protein present.

Next, tests with digestive enzymes were performed. The addition of enzymes that digest proteins and RNA left the agent intact, while enzymes that digest DNA completely destroyed the substance.

Finally, immunological tests involving centrifugation and electrophoresis were performed, which also showed that proteins and polysaccharides weren’t present, but that DNA was.

Once Avery was satisfied with the results of his tests, he began writing a manuscript that explained the experiment. In January of 1944 “Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types” by Oswald T. Avery, Colin M. MacLeod, and Maclyn McCarty was published. Although it may seem that Avery and his colleagues had proven that DNA was the site of the gene, this was not entirely the case. There was still a possibility that, as Avery puts it in the manuscript, “the biological activity of the substance described is not an inherent property of the nucleic acid but is due to minute amounts of some other substance adsorbed to it or so intimately associated with it as to escape detection. . .”

Avery was clearly being very cautious in his conclusions, never stating that he was certain that DNA was the transforming agent. It is possible that his cautiousness with the matter contributed to his lack of attention received. However, there were other reasons why Avery wasn’t given serious attention. As James Watson later stated in 1983:

Both Francis and I had no doubts that DNA was the gene. But most people did. And again, you might say, ‘Why didn’t Avery get the Nobel Prize?’ Because most people didn’t take him seriously. Because you could always argue that his observations were limited to bacteria, or that [the transformation of pneumococcus that he described was caused by] a protein resistant to proteases and that the DNA was just scaffolding.

Although Avery’s manuscript may not have been received with high praise at the time of its publication, it is now considered to be a very thorough account of an expertly accomplished experiment. For more information on DNA, please visit the Race for DNA website. For information on Linus Pauling, a major player in the DNA story, visit the Linus Pauling Online portal.