From the Suitcase: Avery Discovers Genes Are Made of DNA

Few questions stump biologists of all shapes and sizes, from grad students to department heads, like “who discovered DNA?” In part, because it’s usually the wrong question. What most people actually want to know is who demonstrated that genes (the abstract carrier of heritable properties, the “yellow” or “wrinkled”-ness of Mendel’s peas) were made of deoxyribonucleic acid. The answer to the first question is Friedrich Miescher discovered DNA (“nuclein” in his words) while studying, for lack of a better word, pus in the 19th century. The wrong answer to the second question is “Watson & Crick” (the slightly more sophisticated wrong answer to the second question is “Hershey & Chase”).

Behind Door #3 we have Oswald Avery, accompanied by Maclyn McCarty and Colin MacLeod (it’s a big door):

“Biologists have long attempted by chemical means to induce in higher organisms predictable and specific changes which thereafter could be transmitted in series as hereditary characters. Among microorganisms the most striking example of inheritable and specific alterations in cell structure and function that can be experimentally induced and are reproducible under well defined and adequately controlled conditions is the transformation of specific types of Pneumococcus. This phenomenon was first described by Griffith who succeeded in transforming an attenuated and non-encapsulated (R) variant derived from one specific type into fully encapsulated and virulent (S) cells of a heterologous specific type.”

That’s how they introduce the problem tackled in their landmark 1944 JEM paper “STUDIES ON THE CHEMICAL NATURE OF THE SUBSTANCE INDUCING TRANSFORMATION OF PNEUMOCOCCAL TYPES”.  The stated goal is straightforward: to determine which chemical component of bacteria is responsible for conferring the encapsulated, virulent property. I’ve read summaries of this over the years, and seen many textbook illustrations. But here is what the experimental readout actually looked like, on the left are the attenuated, and on the right, the virulent (transformed) bacterial colonies (click on picture to enlarge):

Presentation2

The conclusion, after an epic series of biochemical purification experiments was clear:

“A desoxyribonucleic acid fraction has been isolated from Type III pneumoeocci which is capable of transforming unencapsulated R variants derived from Pneumococcus Type II into fully encapsulated Type III cells.”

Avery and his collaborators could also see that their results posed an important new question: which chemical properties conferred on DNA the ability to transmit information?

“If it is ultimately proved beyond reasonable doubt that the transforming activity of the material described is actually an inherent property of the nucleic acid, one must still account on a chemical basis for the biological specificity of its action.”

The need to answer this question was the starting shot in the race to understand the structure of DNA- the race that Watson and Crick did win, in 1953. In fact Avery et al‘s problem very neatly sets up the double helix paper’s famously coy conclusion:

“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.”

Alfred Hershey and Martha Chase? They did beautiful work in 1952. But it was an atomic age confirmation of Avery, MacLeod, and McCarty’s 1944 classic.

Bibliography:

Avery, O.T., MacLeod, C.M., and McCarty, M. 1944. Studies on the chemical nature of the substance inducing transformation of Pneumococcal types. J. Exp. Med., 79:137. 

Hershey, A.D and Chase, M. 1952. Independent functions of viral protein and nucleic acid in growth of bacteriophage. J. Gen. Physiol. 36:39.

Watson, J.D. and Crick, F.H.C. 1953. A structure for deoxyribose nucleic acid. Nature. 171:737. 

The First Five Pages: a Conversation With Matthew Cobb

“Now what you’ve got to do is get another paper like that Avery paper” is Matthew Cobb’s advice to Journal Experimental Medicine, referring to the series of JEM papers where Oswald Avery and his team demonstrated that genes were made of DNA. He’s just got one small, but pertinent, question: “What would it be on? What would be the topic?” Ay, there’s the rub…

Cobb’s own work focuses on olfaction in maggots, and somehow he also finds the time to write books on the Second World War. His latest book, Life’s Greatest Secret has been shortlisted for the Royal Society Winton Prize, and received rave reviews. Dr Cobb discussed with us the inspiration behind his books, how a scientist gets a book published, and the challenges of studying the history of science.

Cobb final 1

Illustration by Madalena Parreira.

Why did you write a new history of the golden era of molecular biology?

Matthew Cobb: I could see there was a story there that I could explain to the general reader. By concentrating on the experimental detail, which is something I really know about, I could explain the story in a novel way. That was the motivation. When we learn about this period of scientific history, to the extent that we do learn about it, it’s generally confined to the first five pages in the textbook, that people probably skip over because they are not interested. It’s told in a very linear way, and of course history is only linear when you’re looking back. When things are happening, just like they are now, it’s really confused, and there are lots of interactions. It’s not clear where you’re going, and I wanted to recapture some of that confusion, and some of that groping, which you can see in the words that people use. I found myself using the word “information”, or the word “codon”—I didn’t allow myself to use that word until late 1961, by which point the word had been invented. This was really difficult, to force myself into this straightjacket. But at the same time it was essential to try to explain things in the right way and to show why what now seems obvious was once unknown.

So Life’s Greatest Secret focuses on the experimental work of cracking the code?

MC: As well as reading memoirs and so on, I read all the original papers—for every chapter. For the Jacob/Monod chapter, this was pretty heavy going, because I have no training in bacterial genetics at all, and their papers are very tough going, if you haven’t had someone take you by the hand and explain it all. I found that very difficult. But it was something I had to go through. I couldn’t explain it to the general reader if I couldn’t completely understand it myself. So the first task was to read the papers without knowing what was going to come next, just read them blank and imagine “OK, this is the frontier of knowledge now, how can I explain this, and what are the insights that are here, what’s novel, and where are the influences, where have these ideas about come from?”

You’re a working scientist, with a research group at Manchester University. How do you find the time to write about the history of science for a general audience?

MC: I don’t watch TV. That’s the simple answer.

Not even The Wire?

MC: [Laughs]  I watched The Wire. So, occasionally, boxsets.

Your first book was The Egg and Sperm Race. You mentioned in an interview with  American Scientist that what prompted the writing of that book was finding a copy of Swammerdam’s work in Paris.

MC: That’s right, that’s how that all started

The topics of the two books are separated by several centuries. Was there a common challenge in writing The Egg and Sperm Race (Generation in the US) and Life’s Greatest Secret?

The biggest challenge in writing the books, Life’s Greatest Secret and Generation, has been trying to put myself back in time, not to use words and concepts that weren’t appropriate for the time. So, in The Egg and Sperm Race, it was any idea of inheritance. I couldn’t write about inheritance because I was writing about the 17th century and although most people find this very surprising, the idea of there being something called heredity didn’t come about until the 1830s. So it was just this concept that didn’t exist. Yes, you could inherit debts, basically, or if you were lucky, a lot of money. But you couldn’t inherit a disease. ‘Heredity’ took on a biological meaning only in the 1830s.

Similarly, in Life’s Greatest Secret, the biggest difficulty was not talking about information, or code, at a time when those words weren’t appropriate. Genetic information, which is really the whole heart of the book, this idea that everybody shares today. If you explain to somebody what’s in a gene, if you explain to students, ultimately, it’s information. It’s information in the DNA sequence that’s going to produce a protein. That idea came about in a very particular time. In fact, we can date it very, very clearly to the end of May 1953, when Watson & Crick published their second paper in Nature. Not the one on the double helix structure, but another paper, published six weeks later, on what they called the genetical implications of that structure. That’s what the book is about. Where those ideas came from, how they sort of popped into Francis Crick’s mind, why he put them down on the page, and what happened to them once he set them into the wild.

Once you had a topic for your first book, how did you find a publisher? 

MC: It depends on what country you are in. If you’re in the UK, or the US, you need a literary agent, and simply by googling names, putting in “scientific literary agents” and your country, you’ll find various people who are appropriate. This wasn’t quite in the days before Google, but I started thinking about writing the book in about 1998…

So you went to AltaVista…

MC: [Laughs]  Not quite. I contacted Steve Jones, the UK science writer and Professor of Genetics at University College, because he’d published a lot of popular books and I wrote him a letter—or maybe an email… We had email. It wasn’t written with a quill pen. I sent him an email that said “who’s your agent?” He replied “Well, I’m not sure my agent is right for you, but this guy is just set up, he used to be the books editor at Nature, he’s called Peter Tallack.” So I sent an email to Peter basically describing what was going to be a rather boring book. I wanted to write a biography of Jan Swammerdam, this amazing 17th century anatomist and entomologist who was one of the men who could lay claim to having discovered that women have eggs. Peter was very nice about it, and he said he thought it was too academic. I said, OK, I’ll carry on writing and maybe I’ll get it published by one of the academic presses.

Then I suddenly came up with this idea of rather than write the biography, why not concentrate on this huge row that broke out in 1672 over who was the first to discover that women have eggs? This was a row that involved Swammerdam, Steno, who most people won’t have heard of, unless they’ve done Geology (he’s the guy that worked out that the different layers in rocks are looking back in time) and a third guy, who people may have heard of, who was Regnier de Graaf. de Graaf won the race in terms of history, because we now call the place where the eggs come from the Graafian follicles. So I sent Peter a paragraph focusing just on this row, this kind of men behaving badly in the middle of the 17th century, and that it happened at the same time as the Dutch Republic was being attacked by the French, so there was a war and a huge scientific row involving the Royal Society… I just sent this kind of sexed up pitch of one paragraph to him and in 10 minutes I got an email back saying “that’s really exciting, let’s do that”.

Then it was a matter of negotiating with various publishers and talking about a pitch. This is a very, very long process of writing a proposal and then the agent sends it round the publishers. You need to get an agent who is interested in your book, in your idea. Peter Tallack’s (my agent) website is Science Factory, and he’s got a very useful set of FAQs explaining what he needs to know about your idea, and why he’d be interested. The same applies to any literary agent, and they really act as a kind of quality control for the publishers. The publishers use them, and are much more likely to look through a book that comes through an agent than one that just turns up. That having been said, there is a very famous set of books about a boy wizard that was written by somebody who just sent it into a publishers and they all ignored it until about the 18th publisher picked it up. An intern had the job of going through what they call the slush pile, the rejected manuscripts, and she said “hey, Harry Potter is going to make a fortune,” and she was right. So, they don’t always know.

The final section of your book is a look at modern developments in Genetics. Where do you think the frontier is now?

MC: With the development of new sequencing techniques and new methods for looking at what the genome is doing in the cell, we can start to understand the incredibly complex network of repressors and activating molecules that are modulating the activity of genes. To really get a grip on this, we will need to build computer models of those processes, so in a way we’re going back to the ideas that Norbert Wiener came up with in the 1940s about control systems and cybernetics—which is another theme in my book. These ideas are informing systems biology and how genes and their products interact in the cell. I think ultimately it is going to be mind-bogglingly complicated. Which is kind of what we know, because the link between genotype and phenotype, unless it is something simple like eye color, is amazingly complicated. We know that it’s affected by environment and other genes and so on. We’re now beginning to understand how that occurs in molecular terms, and that’s where new models and computer models can be used and then tested by very delicate experimentation in a wide range of organisms, going beyond the traditional ‘models’ into organisms with interesting ecologies that illustrate important facets of evolution. That’s the future.

The Weekend Suitcase

“Today we can begin to understand the cell at a systems level, thanks to the enormous amount of available data that allows us to develop machine learning algorithms to detect patterns that even the brightest mind could not identify.” Stefano Bertuzzi at the American Society of Cell Biology on what cell biologists can learn from Google Cars.

In the past? “Drainage and sanitation projects in the 19th century eliminated many mosquito breeding grounds, but epidemics continued. The last yellow fever outbreak in the USA hit New Orleans in 1905, killing nearly 1,000 people. Malaria was even more difficult to eradicate, stubbornly remaining in pockets of the South into the 1940s and 1950s. The Centers for Disease Control and Prevention was founded in 1946 specifically to combat this problem, which is why their headquarters are in Atlanta: it was then the heart of malaria country.” Are climate change and poverty turning the US in to a “tropical diseases” hotspot, asks Carrie Arnold at Mosaic Science.

“Thus began a fateful test of wills. Merrell responded. Dr. Kelsey wanted more. Merrell complained to Dr. Kelsey’s bosses, calling her a petty bureaucrat. She persisted. On it went. But by late 1961, the terrible evidence was pouring in. The drug — better known by its generic name, thalidomide — was causing thousands of babies in Europe, Britain, Canada and the Middle East to be born with flipperlike arms and legs and other defects.” New York Times obituary for Frances Oldham Kelsey, the FDA official who prevented a tragedy in America, by Robert McFadden.

Bumble says the beginning of the universe was “a lot like this party”. Ah, scientists being awkward at a party. We hadn’t had a derogatory stereotype for nearly 20 seconds. I was getting worried. (…) There may have been more to the video but I must have missed it due to being distracted by the blood leaking from my eyes.” Dean Burnett at The Guardian dissects a video that angered many working scientists on social media.

Lenny Teytelman’s Dear Abby moment: “What is the etiquette for disclosing an anonymous review that you wrote?”

It was a lot more than Aasif Mandvi in tights (though that is pretty good too)- Scientific American compiles a list of Jon Stewarts top 10 science moments.

“Of the three main activities involved in scientific research, thinking, talking, and doing, I much prefer the last and am probably best at it. I am all right at the thinking, but not much good at the talking. “Doing” for a scientist implies doing experiments, and I managed to work in the laboratory as my main occupation from 1940 … until I retired in 1983″. Lara Marks at What Is Biotechnology on the man who taught us how to read genes.

From the Suitcase: Peyton Rous (1910 & 1911).

Peyton Rous had just joined the Rockefeller Institute (forerunner to Rockefeller University) in 1909, when he was visited by a farmer with a sick bird. A chicken’s illness would set Rous on the road to one of oncology’s most important discoveries, the realization that viruses can cause cancer- a finding so controversial that it would take the Nobel committee 50 years to recognize it.

Below are excerpts from Rous’ seminal 1910 and 1911 JEM papers (The Journal of Experimental Medicine’s online archives go back to 1896, and all papers older than 6 months are open access).

“The tumor here reported was found in a barred Plymouth Rock hen of light color and pure blood. It had existed for some two months before the fowl was brought to the laboratory”.

chicken

The patient (from Rous, 1910).

Transmissible, or infectious tumors had been noted before:

“(…) there have since been discovered a number of transmissible new growths of unusual behavior, among them a sarcoma of the dog, transmissible at coitus (Sticker, Ewing), an endemic carcinoma of fishes (Plehn, Pick, Gaylord), and a new growth of hares (yon Dungern and Coca), transplantable to animals of another species.”

In his 1910 paper, Rous created a reliable tumor transplantation system- in the process facing the complex problems of transplantation immunity:

“This was accomplished by the use of fowls of pure blood from the small, intimately related stock in which the growth occurred. Market-bought fowls of similar variety have shown themselves insusceptible, as have fowls of mixed breed, pigeons and guinea-pigs.”

The real leap, however, came in 1911, when Rous showed that an infectious agent was transmitting the cancer:

“A transmissible sarcoma of the chicken has been under observation in this laboratory for the past fourteen months,  and it has assumed of late a special interest because of its extreme malignancy and a tendency to wide-spread metastasis In a careful study of the growth, tests have been made to determine whether it can be transmitted by a filtrate free of the tumor cells. Attempts to so transmit rat, mouse, and dog tumors have never succeeded; and it was supposed that the sarcoma of the fowl would not differ from them in this regard, since it is a typical neoplasm. On the contrary, small quantities of a cell-free filtrate have sufficed to transmit the growth to susceptible fowls.”

The 1911 JEM paper’s method section is essentially a list of filtering and purity verification protocols to exclude bacterial contamination, leading up to Rous’ conclusion that something smaller, an “ultramicroscopic organism” was responsible*:

“The first tendency will be to regard the self-perpetuating agent active in this sarcoma of the fowl as a minute parasitic organism. Analogy with several infectious diseases of man and the lower animals, caused by ultramicroscopic organisms, gives support to this view of the findings, and at present work is being directed to its experimental verification. But an agency of another sort is not out of the question. It is conceivable that a chemical stimulant, elaborated by the neoplastic cells, might cause the tumor in another host and bring about in consequence a further production of the same stimulant. For the moment we have not adopted either hypothesis.”

You can read more on the history of viruses and cancer in Robin Weiss and Peter Vogt, “100 years of Rous sarcoma virus”.

*‘Virus’ had been around since 1892, when Dimitri Ivanovsky coined it to describe the agent causing tobacco mosaic disease, but the term was not yet in wide usage.

Bibliography.

Rous, P. 1910. A transmissible avian neoplasm (sarcoma of the common fowl). J. Exp. Med. 12:696705

Rous, P. 1911. A sarcoma of the fowl transmissible by an agent separable from the tumor cells. J. Exp. Med. 13:397411.

From Aquarium to Bedside: A Talk With Leonard Zon

In the midst of running a very busy lab (“three hundred thousand fish”), treating patients (“I see pediatric hematology patients and adult oncology patients”), creating two companies (Fate Therapeutics and Scholar Rock), and playing the shofar, Dr Leonard Zon found time to talk to JEM. Dr Zon’s fascination in college with hormonal regulation of blood cell development led to over three decades of groundbreaking work, from identifying GATA-1 as a key regulator of erythrocyte development, to his pioneering role in making zebrafish one of the dominant models in hematopoiesis research.

Dr Zon has recently published in JEM on how the nucleoside adenosine promotes hematopoietic development, which together with two other papers in the same issue (one of which he also co-authored) help understand how blood flow in the developing embryo regulates the emergence of hematopoietic stem cells.

A natural storyteller, Dr Zon gave us enough material for more than one post. Enjoy part I.

lz1(1)

Illustration by Madalena Parreira.

How did you get involved in basic research?

LZ I went to Jefferson Medical College and I thought I wanted to be a community pediatrician. I had done research all during college, but I didn’t know that I wanted to do research. Then I went to a lecture by Allan Erslev. Now, Allan Esrlev was the person who discovered erythropoietin. He did the seminal experiment, where he took anemic rabbit sera and shot it into a bunny that was normal, and that bunny developed polycythemia. I was so blown away that hormones could actually control blood cells, and blood cell development, that I thought this is what I want to go into. So I set up a long career of examining cell specific signals and cell fate decisions. That has really captivated my thoughts for 30, 35 years.

Then I came to Harvard to do my clinical training and during my fellowship we had the opportunity to work in a basic science lab, and so I joined Stu Orkin’s laboratory. It was really there that I felt like I got to a place that I really enjoyed, and just loved the intellectual debates and curiosity that went on. So it really kind of hit its stride when I was a fellow. At that point, I had cloned this transcription factor called GATA1, which is required for red blood cell development. It binds to every red blood cell gene and activates it. So that was an exciting time, and that let me ultimately set up my own lab.

Tell us a bit about your recent work on zebrafish hematopoiesis in JEM.

LZ This was a really nice paper, and I’m very happy that we were able to publish it with all the other papers. It really is a very good group of papers together, that I think complement each other. We had found that adenosine worked in a screen looking for engraftment in the caudal hematopoietic territory, which is the fetal liver equivalent (in zebrafish, ed. note). This chemical had also come up in another screen and so it seemed something that was very biologically active, and we really pursued it. A number of adenosine analogs increased the engraftment of the stem cells in this “fetal liver.”

We saw that in the developing aorta, the adenosine analogs really regulated hematopoietic stem cell and progenitor formation. We could use an antagonist to the adenosine pathway and also show that this would block the production of the stem cells. We were able to figure out that adenosine worked through a particular receptor, the A2B receptor. Then we showed that what happens is that you get more budding events if you give a receptor agonist. We could do this with live imaging using confocal and actually watch these things happen. We could also use an antisense approach, or morpholino, approach to the receptor. When you knockdown the receptor, the budding events happened, but then the cells started to die, and they would kind of burst. It meant that the pathway was not only sufficient to induce stem cells, but was also required, at a certain level. Together with George Daley’s lab, we were able to demonstrate that in mouse AGM cultures, as well as embryonic stem cell cultures, activation of this pathway promoted hematopoietic development. That was nice, that the process was conserved.

We went on to show a little about the mechanism. There’s a chemokine, CXCL8, also known as IL8, and what we realized is that adenosine increased the levels of IL8. We found a mutant for IL8, and it had defects in the stem cells in the aorta, particularly through these budding events. The activation of the pathway seems to go through cyclic AMP/PKA. Basically, you have this activation of the receptor, you increase cyclic AMP in the endothelial cell, which leads to the production of growth factors, which would include IL8. This has an impact on the budding event, particularly getting this emerging hematopoietic stem cell, or progenitor cell.

That story was very complimentary to other stories that you published*, the first one being blood flow, and how blood flow initiates this process. George and I had both had papers on that. But we’re able to demonstrate that one of the major activities of the blood flow was actually prostaglandin, and that could also activate a cyclic AMP pathway, and be able to stimulate the production of the blood stem cells. Also, George had been working on CREB as a target gene, particularly as it regulated some of the BMP responsiveness in this region. We were able to look at that and show that this pathway probably also involves the activation of CREB. The stories together really form a very nice unit and show that there are these activators that are triggered by a variety of environmental events including the blood flow, and you have factors like adenosine and prostaglandins that stimulate the initiation of this stem cell budding.

*Ed. note David Traver penned an open-access summary of the three papers.

Another fascinating story is your work on PGE2, where you went from zebrafish to mouse, to clinical trials.

LZ We’re in phase II trials right now. We’re in the midst of testing fifty patients. This was a fantastic story, and I have to give a lot of credit to Trista North and Wolfram Goessling, who were doing the work in the laboratory. They ended up doing a chemical screen to look for inducers of stem cell number in the developing aorta, which is where all vertebrates form their blood stem cells. We found 35 chemicals that can increase blood stem cells. This prostaglandin was definitely the big winner. There was a huge increase in the number of stem cells in the aorta. So we went ahead to see if it was therapeutically useful. We used the mouse competitive transplant assay as a method to see if it would work. We treated marrow with prostaglandin and then competed against untreated marrow that was genetically marked. What we saw was a fourfold increase in the number of stem cells that engrafted if the stem cells had been treated just for two hours with prostaglandin.

Then we took human cord blood. Cord blood is very interesting because the cord blood stem cell transplants work really well, but there are very few stem cells per cord. The current standard of care is to give two cord blood’s worth of stem cells. If just one cord is given, you don’t have an absolute engraftment, so doubling the dose of stem cells gives you a pretty good significant rate of engraftment. The problem with this is that if you put two immune systems in a third person then they start fighting each other. One of the cord bloods will win, in terms of an engraftment, over time. Most people think it would be nicer if you could treat with one cord blood and have it amplified in some way. There are also thoughts about bringing a lot of cord blood that’s been stored that has an inadequate dose of stem cells. If you cold goose those cords up with some drugs it might be useful to actually transplant those—which might be better matched to a patient.

So, we treated cord blood with prostaglandin, transplanted it into immune-deficient mice, and we saw more mice had human blood, and they were more chimeric. Then we decided to do a clinical trial, which was very exciting. I definitely think that in my life, it is the most interesting thing we’ve done. Some of the experiments that we had to do to get through the FDA were extremely boring. At one point we had to change our buffers, because we were using mouse buffers, and we needed to switch to human buffers. I didn’t think that was a big deal. They told me we had to redo all of our preclinical studies in the new buffer.

Thousands of dollars later, the result was basically the same, but that allowed us to go to the FDA and get approval. Then we were able to do this trial, and in the trial we treated 12 patients who had leukemia. They were getting two cord bloods as standard of care, and we basically repeated the mouse experiment in the human by treating one of the cord bloods with the prostaglandin. What we saw was in 10 out of 12, the treated cord was the one that engrafted, and also the treated cord had early engraftment with neutrophils—early recovery, I should say, with neutrophils and platelets about four and a half days earlier.

That’s been really great. We published that in the journal Blood, and then we went on to do a phase II clinical trial. I should say that I started a company as result of this, called Fate Therapeutics, and they’re actually running the phase II trial. I don’t have as much to do with it as I did at the initiation of the first trial. But it is to treat six patients and see how it works. So it is an interesting time, going to phase II trials now for pediatric patients, for inborn errors of metabolism. There are actually three trials that are going on.