Avery, MacLeod, and McCarty are seriously under-acknowledged… They figured out that genetic info’s in DNA and they describe it in such a beautiful way! In last week’s Bri*fing we looked at the “Research” flavor of lab group meetings – and today I want to introduce you to the other kind of group meeting we have (our lab alternates weeks)- JOURNAL CLUB!

Labs often have “journal club” where one person presents the findings of a paper they found interesting to the rest of the lab and they discuss it (our lab has a rotating schedule where 1 week someone presents their own research (I’m up in a couple weeks…) and the next week someone presents a journal article. 

This week we had “journal club” and we talked about a pretty technical paper so I’m not going to try to explain in this post – instead I’m going to use one of the classics – I love classic papers because they’re often so purposeful and elegant…

Today I want to tell you about a classic paper, “Studies on the chemical nature of the substance inducing transformation of pneumococcal types. Induction of transformation by a desoxyribonucleic acid fraction isolated from Pneumococcus type III” – published in the Journal of Experimental Medicine in 1944 by a group of scientists working at the Rockefeller Institute – Oswald Avery, Colin MacLeod, and Maclyn McCarty.

Instead of focusing on the science they did, today I’m mostly going to focus on how they tell others about it in their article. But here’s an overview of their work – which is really cool and definitely under-appreciated 

We looked at a classic set of experiments – the Hershey-Chase experiments (blender ring a bell?), Those experiments used bacteria-infecting viruses (“phages”) with radioactively-labeled protein or DNA to infect bacteria and tracked whether the phages injected protein or DNA. (The blender came in because they used it to shear off the phage parts that where just docked on the bacteria, not inside). These experiments are often credited with showing that hereditary (pass-on-able) genetic information is stored in DNA, not protein. And the Hershey-Chase experiments did help show that – but, perhaps more significantly, they helped convince the wider scientific community of this

You see – Avery’s lab had actually done an experiment showing this – but people hadn’t really accepted it for a couple reasons – one was that they didn’t believe DNA had enough diversity to hold such diverse information and less was known about DNA then. And another was that Avery was doing his research on bacteria with the readout being “shape-changing” and there wasn’t any blender-ing to make it “glamorous” 

Avery was studying a type of bacteria that caused pneumonia, Pneumococcus. He built upon some findings from another scientist, Fredrick Griffith. Griffith had found a strain of Pneumococcus bacteria (Pneumococcus Type II) that, thanks to a polysaccharide (many-sugar-containing) capsule that formed nice smooth colonies on a plate (so he called it the S strain) and was virulent (disease-causing) – if he injected mice with it, the mice got sick & died. Unless he heat-inactivated the S bacteria first.

But Pneumococcus Type III R form formed Rough colonies when plated (cuz they don’t have that capsule) and mice didn’t seem to mind getting injected with it. But if you mixed heat-killed S with R the R bacteria (rough colonies, non-virulent) could transform into the S form (smooth colonies, mouse-killing). So if he injected mice with it they’d get sick & die & he could even isolate live S-like bacteria from the dead mice. 

We now know that this is because bacteria can take in foreign DNA through a process called transformation (we can make them do this in the lab to stick in protein-making-instructions housed in plasmids, but they also do it in the wild). But at this time, scientists didn’t know that it was DNA getting transferred. And most people had their bets on protein. 

Avery set out to figure out what the “transforming principle” was made out of – he did this by repeating the experiments but selectively destroying the polysaccharides (that sugary coat), lipids (fatty membranes, etc.), protein, DNA, & RNA components and seeing when the bacteria didn’t transform. 

Now I want to walk you through their actual paper. There are several key components of a scientific paper. Here’s a brief overview, and in the pics I will point out some of these features in the actual article. The actual layouts of papers and section names differ from journal to journal. But they usually contain these core components…

Abstract – this is like a book’s “blurb” but with major spoilers. It tells the potential reader what the paper’s about – what’s the question they were looking at & why; what did they do to answer it; and what were their key takeaways. Some papers do “graphical abstracts” which, as I’m guessing you could guess – I love! Graphics make me giddy – go figure!

Background – What was known about the topic beforehand? What was missing? Why should we care?

Here Avery gives Griffith – and scientists who followed up on his work – their due credit. He tells us about Griffith’s discovery of the “transforming principle” that could change harmless bacteria into harmful ones – and how other scientists had done further experiments on it and with other organisms, but that the chemical makeup of the transforming substance was still a mystery. 

The organization of the rest of it can vary – some put methods here, others put them at the end – and if they’re really detailed, sometimes there are extended methods in the “supplementary information”

Methods – What techniques did you use and how – *specifically* – did you carry them out – details – give us details! The details should allow someone to recreate the experiment. One of the things I hate is that sometimes, for some “methods” authors will say “we did it like X et al.” and then refer you to “X et al.”;s paper which says we did it like “Y et al.” and send you to another paper – and pretty soon you end up at a paper from the 50s that isn’t even available online…

Some papers call it “Materials and Methods” because it often tells you what materials they used & where they bought them from (or how they made them).

Avery, in great detail (as should be done but isn’t always) describes how they prepared the different materials and the different tests they carried out to try to characterize the “transforming principle” – he designed a “transforming test” involving a “reaction system” including 

  1. nutrient broth
  2. serum or serous fluid (containing 
  3. strain of R Pneumococcus (R36A)
  4. extraction, purification, & chemical nature of the transforming principle

You can practically feel their pain in the sentence: “Each constituent of this system presented problems which required clarification before it was possible to obtain consistent and reproducible results.”

They needed a method for measuring “transforming activity” – the serum in the growth medium contains anti-R antibodies which cause R cells to “agglutinate” during growth -> they clump up & settle at the bottom of the tube, leaving a clear liquid “supernatant” above. But S cells aren’t affected by anti-R antibodies, and there aren’t anti-S antibodies in the serum, so the S cells can grow happily all throughout the medium.

So if you grow normal R you get clumps & clear liquid. If you add the transforming principle the R turns to S and you stop being clumpy. So, in order to figure out what’s required for turning R to S you can add S that’s been treated with different things to destroy different components of it (like its proteins, DNA, or RNA). Only when you’ve destroyed the important thing will you stay clumpy. This is just a “tentative” result – so they also plate the cultures on blood agar for closer examination & positive ID. 

They destroyed proteins (“deproteinized”) S using the “chloroform method” – this is one of those cases where they refer you to a mother paper to find out how they actually did it: “-The solution is then deproteinized by the chloroform method described by Sevag (12)”

They destroyed the capsular polysaccharide (that sugary coat that makes S Smooth) using “a purified preparation of the bacterial enzyme capable of hydrolyzing the Type III capsular polysaccharide” – and then they had to re-chloroform it to destroy that enzyme 😛 

Then they purified it and purified it and purified it… They did this using alcohol fractionation. They use alcohol fractionation and describe the active material as separating “out in the form of fibrous strands that wind themselves around the stirring rod” – if you’ve ever done a DNA extraction from fruit or wheat germ, etc. this might sound familiar! 

Enzymatic Analysis – recombinant proteins weren’t available back then (no proteins made-to-order by that whole clone gene into plasmid -> stick plasmid into cells -> get cells to make that protein thing). So it was a lot harder to get pure proteins because they had to figure out ways to extract them from their natural homes. So they used a number of “crude” (unpure but active) and crystalline (pure) enzymes (the authors thank John Northrop & M. Kunitz for donating samples of purified trypsin & chymotrypsin (protein-chewers) & ribonuclease(RNA-chewer))

Results – What did you find out? This isn’t the place for deep analysis – instead it’s the “show me the data and I’ll decide” portion -it’s meant for presenting the findings, so it usually has the bulk of the figures. You might find figures in the other sections but those figures are probably either to give you an overview of the system being studied & it’s place in the bigger picture of things (in the background section) or a proposed model for what’s going on (in the discussion)

In their results section, they have several tables. 

In addition to those experiments looking at how the substances acted, they also wanted to look at what they’re made up of – in the section “Elementary Chemical Analysis” they describe how they analyzed the amount of nitrogen, phosphorus, carbon, & hydrogen in the active material and compared it to what they’d expect for DNA – and they find that the results are really consistent with it (see Table I) – looks like they’ve probably got some pretty pure DNA preps!

They treated the transforming principle with various “crude enzyme preparations” Only when they added an enzyme that could chew up (depolymerize) DNA did they lose transforming potential. 

In Table III & Chart 1 they describe the results of an experiment where they tracked inactivation of the DNAse compared to inactivation of the transforming principle

Dog serum & rabbit serum both contain a DNAse but the DNAse in dog serum gets destroyed easier – half an hour at 60°C kills it – but rabbit serum’s can withstand that – but not 30 min if you up the temp to 65. And, consistent with DNA being the transforming principle, the bacteria stayed S when treated with 60-C treated rabbit sera (DNAse active, so S DNA destroyed) – but got transformed to R when treated with the 65-C treated rabbit sera (DNAse dead, S DNA transformable). 

“Physiochemical Studies” section – here they used a technique called  analytical centrifugation – you might remember this from the Messelson-Stahl experiments – basically you spin things really fast and heavier things sink further faster and you can use this to separate molecules by size. They got a single, sharp boundary providing further proof that their product was pure, etc.

Quantitative Determination of Biological Activity – here  (shown in Table IV) they titrate the transforming activity – basically they dilute it over and over to see how much is needed to cause transformation – their finding: 1 part in 600,000,000! 

Then it’s discussion time!

Discussion – Here the authors try to tie it all together and fit it into the larger scheme of things. And if there are contradictions between this paper’s findings and other paper’s findings, they can discuss why they think this could be. 

They connect their results back to past results & how they’ve added to them (this is the 1st time transformation has been artificially induced in vitro (in a tube) by a “chemically defined substance” – basically people could induce transformation before by injecting stuff, but they didn’t know exactly what was in that stuff – but these authors show that they can do it with just DNA. 

And they hint at the broader significance – acknowledging that, while they’ve only shown it’s DNA in this one example, it likely has a lot wider-ranging implications. And they basically say – this is so cool – you stick in one type of chemical (DNA) & get it to make another type of chemical (that polysaccharide coat) – they couch their excitement in the phrase “equally striking” but you gotta believe they thought it was pretty cool 🙂 

In the discussion and/or conclusion sections, authors also address any outstanding questions (e.g. we still don’t know *how* DNA does it) & propose alternative theories (e.g. the DNA might not be entirely pure and there could be a tiny bit of something else in there tricking us). 

Authors also must account for shortcomings & gaps in their experiments (often at the request of reviewing editors).In this paper, for example, they write: “Admittedly there are many phases of the problem of transformation that require further study and many questions that remain unanswered largely because of technical difficulties.” 

“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 ceils.”

Conclusion – Here the authors try to tie it all together.

The conclusion of this paper is short and sweet: “The evidence presented supports the belief that a nucleic acid of the desoxyribose type is the fundamental unit of the transforming principle of Pneumococcus Type III.”

Then comes the Bibliography or References or Works Cited part where they list the papers they cited in the article so you can go track them down yourself. 

Papers these days often have “Supplemental Materials” which can include additional figures, data sets, extended methods, etc.

A couple weeks ago I talked about scientific conferences – they’re awesome when you get to go to them, but you can’t attend them all. The main way in which scientists communicate their findings with people around the world is through journal articles. It’s hard to keep up, so I schedule “alerts” that email me when papers with key words or by certain authors get published so I don’t miss them. I also frequently check the websites of journals that are relevant to me.

This post is part of my weekly “broadcasts from the bench” for The International Union of Biochemistry and Molecular Biology (@theIUBMB). 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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