Data storage fads come and go – from stone etchings to papyrus scrolls, floppy discs to magnetic tape – but when it comes to the genetic information encoding all the proteins and other things your cells need to make, there’s one data storage form that’s been consistently used by “all” organisms since almost the beginning of life itself – DNA! But, despite this long history, the fact that DNA is the source of hereditary information is actually a very recent discovery, which was quite controversial when announced in the 1950s (a lot of scientists thought the info was instead stored in proteins). So how was the protein/DNA debate settled? With a blender, some bacteria, some bacteria-infecting viruses (“phages”) and scientists named Martha Chase and Alfred Hershey.

There are several things I love about this experiment. Firstly, it’s a really elegant experiment that provided key insight into fundamental biochemistry (and a great example of the scientific method at play). Secondly, it was carried out on the same campus I’m a grad student at (Cold Spring Harbor Laboratory). And, last but not least, it was performed by a female scientist who, like last week’s Bri*fing heroine (Eileen Southgate, who traced (by hand) the roundworm C.elegans’ nervous system) was “only” a research assistant at the time she carried out her work (more on Chase later). So Chase-ing last week’s Bri*fing, today I want to tell you about Martha Chase, Alfred Hershey and the classic “Hershey-Chase experiment” (aka the Waring Blender Experiment).  

Most scientists before the mid-1950s believed that genetic information was stored and transmitted through proteins, not nucleic acids (DNA and RNA). This was a sensible hypothesis (educated guess) because, unlike the 4-letter code of nucleic acids, proteins are made up of a larger (20-letter) alphabet of amino acids. And, while the 4 bases (unique parts) of DNA are all fairly similar, the 20 amino acids have very different chemical properties. Many scientists believed that this diversity made proteins the better candidate for genetic storage. But not all scientists were convinced…

Early Evidence for “genes are DNA” camp:In 1928, Frederick Griffith showed that a “transforming principle” could be transferred from virulent, disease-causing, bacteria to harmless, non-virulent bacteria, “transforming” the once benign bacteria into killing machines. more here: http://bit.ly/32aCOEh

A group of scientists at the Rockefeller Institute (Oswald Avery, Colin MacLeod, & Maclyn McCarty), expanded upon the work and, in 1944, announced the results of an experiment that supported the “DNA camp.” They showed that enzymes that destroyed DNA inactivated the “transforming principle,” whereas chemicals that destroyed proteins but not DNA had no effect. more here: http://bit.ly/33mcB5I 

Still, there was wide skepticism. It just didn’t seem possible that the diversity of life on earth could be written in an alphabet that only had 4 letters.

To settle the debate, Martha Chase and her colleague Alfred Hershey at Cold Spring Harbor Laboratory (CSHL) devised an elegant yet powerful experiment that has gone down as one of the greatest molecular biology experiments of all times, often referred to as the “Hershey-Chase experiment” or the “Waring blender experiment.”

In addition to a blender (yep, one just like you might have in your kitchen), or this one we have in our lab for some reason (this isn’t the one they used (which is on display in the library, (although I did get to use Carol Greider’s pipets once…). The blender might have been “normal,” but they also needed some “special” tools for their experiment – they used a special type of virus, called a bacteriophage or “phage” for short, that infects bacteria.

If “phage” sounds familiar it might be because these bacteria-infecting viruses show up a lot in biochemistry because we use bacteria as “factories” for doing things like making DNA & proteins, and phages are designed to do this so they have a lot of the “equipment” we want. We frequently use “pieces” of the phage – like their DNA copying machinery (polymerase). But these scientists used the intact phage.

It was known at the time that, when a phage infects a bacterium, it docks on the bacterium’s surface, then injects “stuff” into the bacterium, while leaving its “shell” outside. This injected “stuff” contains genetic information that tells the bacterium to make more virus. The bacterium follows these instructions, makes more virus, then burst open (lyses), releasing these viruses to infect more bacteria. But what’s in this “stuff”? Proteins, nucleic acids, or both? Chase and Hershey knew that answering this question would help answer the larger question of what genetic information is made up of, so they devised a plan.

Key to the experiment was figuring out how to distinguish between nucleic acids (DNA & RNA) and proteins, both of which are made up of atoms. Atoms are the basic units of elements like carbon (C), hydrogen (H), oxygen (O). and they’re made up of smaller parts called subatomic particles – positively-charged protons & neutral neutrons hang out in a dense central nucleus, and they’re surrounded by a cloud of negatively-charged electrons which whizz around and interact with other atoms.

Elements are defined by their # of protons (e.g. carbon ALWAYS has 6 protons and oxygen ALWAYS has 8), but the # of neutrons & electrons can vary. Since electrons are negatively-charged, if you change the # of electrons, you change the charge, and that can make the atoms act differently. But if you change the # of neutrons, which are neutral, the atom acts the same.

At least from an “outside atom’s perspective” – inside, there might be conflict brewing – if there’s a significant imbalance between the # of protons & the # of neutrons, the “glue” keeping the + protons from repelling each other gets strained, making the nucleus unstable. And it can become stable by letting off radiation, so we call the unstable versions “radioactive isotopes” or “radioisotopes.” More on this at the end, but I don’t want to stray too far from the story.

All the nuclear instability is happening deep within the core of the atom so other molecules don’t see these “inner demons” and thus they interact “normally” with the struggling atom. So we can swap out “normal” atoms for “radioactive isotopes” of those atoms to act as labels – and measure the radiation they give off to track where the labeled things go. Sometimes, we label things after they’re already made – like when I use hot ATP to radiolabel the ends of synthesized RNA molecules. more here: http://bit.ly/2lb0O9U 

But radioactive atoms can also be incorporated during the making process if you use radioactive “building blocks.” If you want to radioactively label the atoms of specific elements in biologically-produced molecules as they’re being made you can by include a radioactive isotope of that element in an organism’s growth media (food). When the organism makes new proteins and nucleic acids, it will incorporate the radioactive isotope into them, “labeling” them. 

But what to label? Carbon (C), Hydrogen (H), & Oxygen (O) are in almost all of the main molecules that biochemists care about (nucleic acids (DNA & RNA), proteins, lipids (fats & oils), etc.) So labeling those atoms would be like taking a highlighter to an entire page – not very helpful. We need something more specific to different types of molecules. So what other elements do we find in biochemical “macromolecules”? A few common ones are Nitrogen (N), Sulfur (S), & Phosphorus (P). 

Hershey and Chase wanted to tell apart DNA & proteins. Those both have nitrogen, so check that off the list. But what about sulfur & phosphorus?

You’ll find P in places like RNA & DNA (where it’s in the generic backbone of every letter (nucleotide)), but none of the protein letters (amino acids) have phosphorus in them (although phosphorus *can* get incorporated into proteins after they’re made when proteins called kinases take off part of an RNA letter (ATP) and stick a phosphate group on them – this phosphorylation can change the protein’s shape & activity, But that phosphate-adding only happens sometimes to some molecules and after they’re made.) more here: http://bit.ly/2lOgXSN

So we can use radioactive P to label nucleic acids, but not proteins, as they’re being made. How to label proteins? Turn to sulfur. None of the DNA or RNA letters have sulfur, and most protein letters don’t either – but a couple – methionine (Met, M) & cysteine (Cys, C) do.

So – nucleic acids and amino acids both contain carbon, nitrogen, and oxygen, but nucleic acids also contain phosphorus while amino acids don’t. And some amino acids contain sulfur, while no nucleic acids do. Therefore, radioactive phosphorus can be used to selectively label nucleic acids, while radioactive sulfur can be used to selectively label proteins.

Chase & Hershey grew bacteria in growth media containing either radioactive phosphorus (P-32) or radioactive sulfur (S-35) and infected these bacteria with a phage called T2. The bacteria in both media made more of the T2 phage, but the T2 produced by the bacteria in P-32 media had radiolabeled nucleic acids while the T2 produced by the bacteria in S-35 had radiolabeled proteins.

Chase & Hershey isolated these labeled viruses and used them to infect bacteria that were grown in normal media. They allowed the T2 to dock on the bacteria and inject the mysterious “stuff.” Then, before the bacteria lysed (broke open), they used a blender to shear the “shells” of the T2 off of the bacterial cells and centrifuged the resultant mixture to separate the heavier bacteria (now containing the T2’s genetic “stuff”) from the lighter T2 “shells.” They then measured how much radioactivity was in each portion.

When planning an experiment, it’s important to think about what results you would expect in different cases, so let’s think about this for a minute. We have 2 experimental conditions, labeled protein and labeled DNA, and for each of these we’re comparing radioactivity in 2 populations (T2 “shells” and bacteria). We have 2 main hypotheses (“genetic information is made up of protein” versus “genetic information is made up of nucleic acid”). (Of course, there is also the possibility that the “stuff” contains both, but we’re going to ignore that here for simplicity).

So, what did they find? When they labeled the nucleic acid, almost all of the radioactivity was in the bacterial portion; whereas, when they labeled the protein, almost all of the radioactivity was in the T2 portion. This told them that the “stuff” being injected into the bacteria contained nucleic acids, but NOT proteins! And, since this “stuff” held the T2’s genetic information, this information is made up of nucleic acids, NOT proteins

As we know now, DNA stores information in “words” of three consecutive bases (termed codons) that code for one amino acid, thus providing the diversity required for storing complex information. Furthermore, each nucleic acid base is complementary to another base, so the information can be easily copied and transmitted. These properties make nucleic acid ideal for the job (in fact, with the “big data” revolution generating enormous quantities of data, scientists are currently working on using synthetic DNA to store some of it!).

The experimental results weren’t exactly this cut-and-dry (for instance, ~20% of the radiolabeled sulfur ended up in the “cell portion” (likely do to protein that resisted getting sheared off by blending) and ~15% of the radiolabeled phosphorus ended up with the “shell portion” (likely due to lysing and/or viruses that didn’t get a chance to infect yet). So there were still some doubters. 

But this was “just” one (important) set of experiments that added a piece to the puzzle of figuring out what genetic info’s made of – in addition to findings from work by other groups of scientists, Hershey and Chase did many more experiments of their own, described in their same classic 1952 paper, “Independent functions of viral protein and nucleic acid in growth of bacteriophage” (freely available for your reading pleasure http://bit.ly/39TMQNy )

For example, they confirmed findings by another scientist, Thomas Anderson, that phages had DNA inside a protein coat. And they showed you could separate the protein & DNA parts & that the protein-containing parts could stick to bacteria (adsorb to bacterial membranes) but the DNA parts couldn’t. And that the DNA goes inside the bacteria. And that the protein coat protected the DNA from DNA chewers (DNAses) – if you add DNAse to released DNA it gets degraded but when it’s inside an intact phage it’s fine. 

Yeah, they did a lot… And a lot of that was inspired by the work of other scientists – scientific communication & collaboration for the win! Science is done in steps, and those experiments and ones done by fellow scientists, were also really important steps, but they didn’t use blenders so there’s less “wow” factor for history to remember. 

Speaking of remembering, because the work played such a large role in establishing DNA as the source of genetic information, the experiment won Hershey the Nobel Prize in 1969 (he shared it with Salvador Luria and Max Delbrück “for their discoveries concerning the replication mechanism and the genetic structure of viruses.” Martha Chase was not included, and Hershey didn’t even acknowledge her contributions in his acceptance speech. Maybe if Chase had gotten more credit during her lifetime, we would have more information preserved about her. As it stands, it is incredibly difficult to find information about her, but here’s what I was able to find out…

Martha Cowles Chase was born on November 30, 1927 in Cleveland, Ohio. After receiving a bachelor’s degree from the College of Wooster in 1950, she went to work as a research assistant for Alfred Hershey at CSHL. Martha resigned from CSHL in 1953 and went to work at Oak Ridge National Laboratory and the University of Rochester, returning every summer in the 1950’s for CSHL’s famous “Phage Group” meetings. After almost a decade working as a research assistant, she decided to return to school. She moved to California and began doctoral studies in microbiology at the University of Southern California, receiving a PhD in 1964. A series of setbacks ended her scientific career, and she moved back to Ohio after graduating to live with family. The last decades of her life were marred by a form of dementia affecting her short-term memory. She died of pneumonia in 2003. 

There is little surviving information about the level of “intellectual contribution” Chase played in the experiment, but, given that she was listed as a co-author on the results paper (which was not common practice for research assistants unless they contribute substantially), she is believed to have played a key role throughout the experiment’s design, execution, and interpretation. Some denigrate research assistants and technicians as merely “sets of hands,” but these workers aren’t just manual labor, they are scientists, and when they make significant contributions, they deserve credit. So, to all the other under-appreciated research assistants and technicians who help make science possible! Thank you for your work!

Steps down from soapbox to wrap up post with the promised note on the radioisotopes used…

So, nuclear decay is a way for atoms to stabilize nuclei that have too many or too few neutrons compared to protons. There are a few kinds of radiation. Alpha-decay gives off the equivalent of a Helium atom (2 protons & 2 neutrons), Beta-decay swaps a neutron for a proton (in beta-plus decay) or a proton for a neutron (in beta-minus decay (aka positron emission) and lets off an electron and antineutrino (in beta-plus) or an anti electron & neutrino (in beta-minus) to conserve charge & weird tiny physics stuff. Both P32 & S35 decay through β-minus decay.

Both of those (alpha & beta) involve changing the # of protons, so they change the atom’s identity. But a third type of radiation, gamma-decay, doesn’t give off any physical particles, instead it just releases energy, in the form of radiation – all electromagnetic radiation (EMR) – everything from microwaves to infrared to visible light to ultraviolet (UV) to x-rays to gamma rays is the “same” – little packets of energy (photons) traveling in waves through space  – they just differ in how much energy they have. 

The more energy, the higher the frequency & shorter the wavelength (more up-downs to travel the same distance). Gamma rays are like x-rays on steroids – they’re really high-energy & thus dangerous. Gamma rays can be given off by themselves, but they’re often given off alongside those other element-changing forms of decay. more here: http://bit.ly/radiolabeling

I hope that this article helped you better understand and appreciate the elegance of this groundbreaking experience and I hope that when you think of the Hershey-Chase experiment you will think about Martha Chase!

This post is part of my weekly “broadcasts from the bench” for The International Union of Biochemistry and Molecular Biology. Be sure to follow the IUBMB if you’re interested in biochemistry! They’re a really great international organization for biochemistry.

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0

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