gg at Skulls in the Stars has issued an interesting challenge to science bloggers:  write a blog post about a classic scientific research paper, preferably something pre-WWII.  This is a great idea, and I hope plenty of bloggers take up the challenge.  Reading old papers is great, and I wish that science instructors would utilize them more in their classes.  Not only does it foster an appreciation of the history of science, but it also brings the material to life and makes learning more enjoyable.  Granted, not all old papers are a joy to read, and some are painfully written, but I think they provide the reader with a much deeper understanding of the topic than just learning facts by rote.

Unfortunately, I don’t think I’ll be able to fulfill gg’s challenge to the letter.  My realm of expertise is in biochemistry, biophysics, and molecular biology, and these fields of study only started to take off relatively recently.  Most of the classic papers in these fields come from the 1950s.  Watson and Crick published their paper on the double helical structure of DNA in 1953.  The first protein structure solved by x-ray crystallography was published in 1959. So when I was researching the topic of this blog post, I had a hard time coming up with much before WWII.  Originally I thought I would write about Messelson and Stahl’s 1958 paper on the semiconservative nature of DNA replication.  Their experiment was really quite elegant, and it would be great paper to write about.  But 1958 seemed a bit late, so I searched a bit more and decided on a seminal paper by Oswald T. Avery, Maclyn McCarty, and Colin MacLeod.  Published in 1944, Avery’s paper, Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types, was the first to identify DNA as the molecule that carried genetic information, and became a watershed in the science of genetics.

First, a bit of historical background.  Before Avery and coworkers published their paper, there was very little interest in DNA among biologists.  Relatively little was known about DNA, but early analyses suggested that it was a very simple molecule, at least in terms of its chemical composition.  This view was best represented by the so-called “tetranucleotide hypothesis”, which held that DNA was composed of equal amounts of four nucleotides, adenine, guanine, thymine, and cytosine.  Thus, the prevailing opinion at the time held that DNA was far too simple a molecule to carry genetic information.  Instead, there was much interest in proteins as the chemical identity of genes, which was understandable in light of the tetranucleotide hypothesis.  In contrast to the supposed simplicity of DNA, proteins, composed of varying amounts of 20 amino acids, appeared to possess the chemical diversity required to function as genes.

Avery et al. came into the picture investigating a process called transformation.  In transformation, cells undergo a genetic change following the uptake of foreign DNA (of course, this wasn’t known prior to the work of Avery and coworkers).  Transformation was discovered by Robert Griffith in 1928, shortly before Avery’s group began their work.  In a widely renowned experiment, Griffith demonstrated that bacteria are capable of transferring genetic information. Griffith worked with two strains of the bacteria pneumococcus, named according to the appearance the colonies grown from each strain.  The S-strain possessed a polysaccharide coat and formed large, smooth colonies.  The R-strain did not have a polysaccharide covering, and grew in small, rough colonies.  In addition to influencing colony appearance, the polysaccharide shell also protected the S-strain from the host immune response.  Griffith noticed that mice injected with the S-strain eventually became ill and died, while mice injected with the R-strain suffered no ill effects.  He also noted that S-strain pneumococci that had been killed by heat caused no harm.  But Griffith’s breakthrough came when he found that mice injected with a mixture of live R-strain and heat-killed S-strain died from infection.  Furthermore, he was able to isolate live S-strain bacteria from these dead mice.  From this, Griffith concluded that the heat-killed cells contained a “transforming principle” capable of changing the harmless R-strain bacteria into the pathogenic S-strain.

Avery, McCarty, and MacLeod set out to isolate and identify Griffith’s “transforming principle”.  However, rather than using mice, Avery’s group developed an in vitro system that allowed them to monitor transformation.  In this system, R-strain bacteria would be mixed with extracts from S-strain cells and grown in a nutrient broth containing anti-R-strain antibodies.  During growth, the anti-R antibodies cause the R-strain cells to aggregate together and eventually settle at the bottom of the test tube, leaving the nutrient broth clear.  If transformation occurs, the resulting smooth bacteria, unaffected by the antibodies, remain free to diffuse as they grow, producing a cloudy solution.  As a result, Avery and coworkers could easily determine if transformation had occurred, with no need to wait for mice to die.

Establishing this system was no easy task.  Avery and MacLeod had begun working on in vitro transformation in the early 1930s, and it was nearly 10 years before they had the kinks worked out.  Understandably then, Avery begins his landmark paper discussing some of the problems they faced in achieving a consistent transformation procedure.  For example, one difficulty involved the serous fluid they used as a source for antibodies to the R-strain:

It has been found that sera from various animal species, irrespective of their immune properties, contain an enzyme capable of destroying the transforming principle in potent extract. (…) This enzyme is inactivated by heating the serum at 60°-65°C, and sera heated at temperatures known to destroy the enzyme are often rendered effective in the transforming system.

We’ll see later that this difficulty becomes useful for Avery’s group in determining that the transforming priciple is DNA.  I won’t go into detail on the other problems they faced establishing their transformation system, but it is impressive that they were able to overcome them.  Transformation is a common procedure in labs today, but despite all we know today, it can still be a finicky process; it’s not uncommon for transformations to fail for no obvious reason.  So I can’t imagine what it must have been like to work on transformation in the ’30s.  I’ve been doing science just long enough now that I’ve experienced the frustration and dissatisfaction that come with repeated failure.  But to work on the same thing for ten years with limited success?  It must have been maddening.  That Avery’s group was able to slog through the grueling repetitiveness and establish reliable in vitro transformation is simply remarkable, and it is a testament to their skill as scientists and to their patience.

With their transformation system in place, Avery, MacLeod, and McCarty could start their efforts to purify and characterize the transforming principle.  Avery summed up this daunting task in a letter to his brother in 1943:

“The crude extract [from S-strain] is full of capsular polysaccharide, C (somatic) carbohydrate, nucleoproteins, free nucleic acids of both the yeast [RNA] and thymus [DNA] types, lipids and other cell constituents. Try to find in that complex mixture the active principle…Try to isolate and chemically identify the particular [transforming] substance…. Some job-full of heartaches and heartbreaks.”

For several years the group worked to obtain pure transforming principle, and in their paper they report the successful procedure.  The purification process itself provides clues to the chemical nature of the transforming principle.  They began by washing heat-killed S cells to remove the polysaccharide coat and then lysing (breaking open) the cells to extract the intracellular material.  Insoluble debris was then removed by centrifugation, and purification proceeded from the soluble extract.  From this point the process incorporated three key steps:

1. Removal of proteins by chloroform extraction.
2. Enzymatic digestion of remaining polysaccharides.
3. Precipitation of active substance with ethanol.

The resistance of the transforming principle to chloroform extraction had to have been fairly alarming to Avery, considering the prevailing view that proteins were the source of genetic information.  Furthermore, enzymatic digestion was evidence against polysaccharides, and ethanol precipitation was a strike against lipids, as lipids are generally soluble in ethanol.  Things were starting to point to nucleic acids.

With purified transforming principle in hand, Avery’s group began their analysis.  To begin, they noticed that the pure substance gave negative results for two chemical tests for the presence of proteins, while the Dische diphenylamine reaction, used to test for the presence of DNA, produced a strong positive result.  Then, an elementary chemical analysis was performed on the substance to determine its composition.  Avery noticed that the results of this analysis matched very closely with theoretical values for DNA:

Notice that the elemental analysis is further evidence against a protein transforming principle.  Proteins do not contain phosphorous unless they are phosphorylated post-translationally, but even phosphorylated proteins contain only a few phosphorous atoms.  If Avery’s purified transforming principle contained significant amounts of protein, the N/P ratio would be much larger.

Visual inspection of the transforming principle also pointed to DNA.  Avery’s group found that when they precipitated the purified transforming principle with ethanol, it formed a shimmering, fibrous mass that very much resembled DNA isolated from shad sperm by an upstairs colleague, Alfred Mirsky.

With DNA looking like a good candidate for the transforming principle, Avery’s group performed an enzymatic analysis of their substance.  Solutions of pure transforming principle were treated with the enzymes trypsin and chymotrypsin, which digest proteins.  These had no effect on the transforming principle.  Treatment with ribonuclease, an enzyme that breaks down RNA, also had no effect.

In addition to purified enzymes, they also tested extracts from various animal organs for specific enzymatic activity and compared that to the ability of the extract to inactivate the transforming principle:

In the above table, a plus sign indicates the presence of an activity, a minus sign indicates no activity.  As the table shows, only those extracts that could break down purified DNA inactivated the transforming principle.  Phosphatases and esterases had no effect on transformation.

Furthermore, as I mentioned earlier, Avery’s group knew that they could inactivate an enzyme in serous fluid that destroyed the transforming principle by heating the serum at 60°-65°C.  With this in mind, they heated dog and rabbit serum and then tested for DNA depolymerase activity.  They found that, like the ability to prevent transformation, sera heated at 60°-65°C  could not depolymerize DNA. From these enymatic analyses, Avery’s group concluded:

The fact that transforming activity is destroyed only by those preparations containing depolymerase for desoxyribonucleic acid and the further fact that in both instances the enzymes concerned are inactivated at the same temperature..provide additional evidence for the belief that the active principle is a nucleic acid of the desoxyribose type.

All of this evidence led Avery’s group to conclude that DNA was the “fundamental unit of the transforming principle of Pneumococcus”.  The group was keenly aware of the importance of their finding, should it be correct, but they also realized that this conclusion might be controversial to many of their peers, and the paper discusses the result in a cautious tone:

It is, of course, possible that 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. If, however, the biologically active substance isolated in highly purified form as the sodium salt of desoxyribonucleic acid actually proves to be the transforming principle, as the available evidence strongly suggests, then nucleic acids of this type must be regarded not merely as structurally important but as functionally active in determining the biochemical activities and specific characteristics of pneumococcal cells. Assuming that the sodium desoxyribonucleate and the active principle are one and the same substance, then the transformation described represents a change that is chemically induced and specifically directed by a known chemical compound. If the results of the present study on the chemical nature of the transforming principle are confirmed, then nucleic acids must be regarded as possessing biological specificity the chemical basis of which is as yet undetermined.

I would like to say that the Avery paper did not, at that time, definitively establish DNA as genetic material.  Rather, it only showed that DNA was the transforming principle, and there were competing ideas about the mechanism of transformation.  However, many scientists did interpret transformation as a genetic phenomenon, so the genetic implications of the paper were immediately recognized.  That said, the paper did not find immediate acceptance.  In 2003, two years before his death, Maclyn McCarty discussed the reception of his work, saying:

Our findings continued to receive little acceptance for a variety of reasons, the most significant being that the work on the composition of DNA, dating back to its first identification 75 years earlier, had concluded that DNA was too limited in diversity to carry genetic information. Even those biologists who had considered the possibility had dropped the idea, and the prevailing dogma was that if genes are composed of a known substance, it must be protein.

Fortunately, their work did impact some scientists, particularly Erwin Chargaff.  After reading the paper, Chargaff changed the focus of his research to DNA, and subsequently disproved the tetranucleotide hypothesis (see Chargaff’s rules).  Chargaff’s work, and by extension the work of Avery’s group, was of great importance to Watson and Crick as they built their model of the DNA double helix. However, Avery’s finding did not gain full acceptance until the work of others, particularly Hershey and Chase in 1952, and Watson and Crick in 1953, firmly established DNA as the molecule of heredity.  Unfortunately, Oswald Avery died in 1955, before he could receive the Nobel Prize.

References:

Avery, O. T., MacLeod, C. M. & McCarty, M. Studies of the chemical nature of
the substance inducing transformation of pneumococcal types. Induction of
transformation by a desoxyribonucleic acid fraction isolated from
Pneumococcus Type III. J. Exp. Med. 79, 137–158 (1944).

McCarty, M.  Discovering Genes are made of DNA  Nature. 2003 Jan 23;421(6921):406.

Letter from Oswald Avery to Roy Avery, 1943.

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