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Monday, March 24, 2014

Thesis defense - 40th anniversary

Today is the 40th anniversary of my Ph.D. oral defense.1 The event took place in the Department of Biochemical Sciences at Princeton University back in 1974.

It began with a departmental seminar. When the seminar was over I retired with my committee to a small classroom for the oral exam.

I don't remember everyone who was on my committee. My Ph.D. supervisor (Bruce Alberts) was there, as was my second reader, Abe Worcel. I know Uli Laemmli was there and so was Arnie Levine. I'm pretty sure the external member of the committee was Nancy Nossal from NIH in Bethesda, MD (USA). It's a bit of a blur after all these years.

I remember being fairly confident about the exam. After five and a half years I was pretty sure that everyone on my committee wanted to get rid of me and the easiest way to do that was to let me pass. Bruce stood to gain $3000 per year of research money and Uli was going to get back the basement of his house where Ms. Sandwalk and I had been living for the past month.

The toughest questions were from Uli Laemmli, which should not come as a surprise to anyone who knows him. He has this annoying habit of expecting people to understand the basic physics and chemistry behind the biochemical sciences. Fortunately, my inability to answer most of his questions didn't deter him from voting to pass me.

This photograph was taken at a party that evening. I look pretty calm at that point but this may have had a lot to do with the various refreshments that were being served.

The amazing thing about the photograph—as I'm sure you all agree—is how little I've changed since then—apart from a haircut.

Back in those days we didn't spend a lot of time writing a thesis. I started in the middle of January and the entire process of writing and defending took nine weeks. My thesis was bound and delivered to the library about one week after the Ph.D. oral.

The second page of my thesis has only three words on it. It says, "To Leslie Jane." This is Ms. Sandwalk. She really should have her name on the cover 'cause I couldn't have graduated without her. Typing my thesis was only one of her many contributions. There are 257 pages in my thesis and she typed every one. As a matter of fact, she typed them twice, one draft and then the final version.

The figures in my thesis were all hand drawn. I've included one (below) to illustrate what I was doing during those five and a half years.

The Alberts lab was interested in DNA replication during bacteriophage T4 infections of E. coli. We knew that replication was carried out by a complex protein machine that assembled at a replication fork but we didn't know all the players or what they did.

The T4 proteins required for DNA replication were known from genetic studies. The most important genes were genes 30 (ligase), 32 (single-stand DNA binding protein), 41, 43 (DNA polymerase), 44, 45, and 62. The products of the unknown genes were called 41P, 44P, 45P and 62P.

We wanted to purify and characterize those proteins; my target was the product of gene 41, or 41P.

We had a cool assay, developed mostly by a postdoc in the lab named Jack Berry. What we did was to prepare a cell lysate from cells that had been infected by bacteriophage carrying an amber mutation in one of the genes. This lysate could not support DNA synthesis, as measured by incorporation of 32P nucleotides, unless we added back the missing component. This is the basis of an in vitro complementation assay that worked for each of the unknown proteins.

In my case, I used traditional protein purification methods to isolate fractions of proteins and them tested them for activity in the complementation assay. The figure below shows the elution profile of proteins bound to a hydroxylapatite column. The peak centered on fraction 61 is the activity of the complementation assay. It indicates that 41P elutes early as a sharp peak in the elution profile.

The complementation assay doesn't tell us anything about the function of 41-protein, only that it complements an extract that's deficient in 41P. Strictly speaking, it doesn't even tell us that the activity is due to the product of gene 41 since it could be something else that complements in vitro.

Fortunately we had another way of identifying 41P. I started my purification with extracts from 17 liters of infected cells. To this I added extracts from cells that had been labeled with radiaoctive amino acids. One batch was from a wild-type infection where all T4 proteins are labeled with 14C amino acids. The other batch is from an infection with an amber mutation in gene 41. In this case every protein except 41P is labeled with 3H amino acids.

You can adjust the settings on a scintillation counter so they distinguish between 14C and 3H but there's some overlap. The equations for calculating the contribution of each isotope in each window are relatively simple. All you need are good standards to get the distribution. One of the most fun things I did as a graduate student was to write a computer program (in Fortran) that did these calculations automatically and plotted them on a plotter. This was back in the time when computers were housed in large separate buildings and required dozens of people to look after them.

If you look of the elution profile in the figure you'll see there's an excess of 14C over 3H in the same fractions where the complementation activity is located. What this means is that the wild-type extract has a protein at that position that's not found in the am41 extract. It's another way of identifying the product of gene 41.

The double label technique was useful 35 years ago but nobody does it anymore. It was fun while it lasted.

(I never did figure out what 41P did during DNA replication but a few years after I left a postdoc identified 41P as a helicase—an enzyme that unwinds DNA ahead of the replication fork. The enzyme is now called gp41 for "gene product.")

1. This post is an almost identical copy of one that was posted five years ago. You'll probably see another in 2019, and especially 2024.


Leslie Jane Moran said...

I think I should add that this was all typed on a traditional typewriter, before the computer age. If you made an error, or the page was amended in any way, you had to start all over again. It was more like 5 "printings" by the time the pages were worked and reworked. In the end, it was worth it. I'll crack open the champagne tonight :) Perhaps you should invite our lawyer....also got him out of Princeton in a hurry!

Bob Woodham said...

Congratulations on your 40th. It is something to celebrate together.

Leslie Jane Moran said...

BOB!!! Wow! Now there's a blast from the past. Perhaps we will have to share our 47th. or 48th. We may be in Vancouver in November. Dinner??

Piotr Gąsiorowski said...

I wrote mine during the transition period. I typed the first chapter on an old Olivetti, and then re-typed in using an early version of Word for Windows, learning to use it as I went along.

Bill said...

I hired a typist to transcribe my handwritten thesis. Alas, she had a nervous breakdown and I found out only weeks before my oral that she had only finished about a quarter. I had to buy a typewriter and finish the rest myself, and correct her mistakes. (To be fair, I paid her the agreed amount, regardless.) Those were the days.

Earlier I did work on analyzing dipeptide fragments from GC-MS using artificial intelligence to interpret the mass spectra.

Now, people are, like, oh, well, do a DNA analysis - put it in a machine. I guess it was cool to be working in the pre-machine age!

Hey, creationists, tell us about your grad student days! Hello ...

Sean Boyle said...

Wow I'm digging your look back then! It seems everyone from that era looked so cool. Reminds me of my father and his brother, the curls with the mustache is such a classic 70's look.

Bob Woodham said...

Sorry to have missed the NHS reunion last May. Do let me know when you both are visiting Vancouver. Dinner would be great! It's been a while for the class of '67.

SRM said...

Nice to see the use of hydroxyapatite, it was one of mediums I used to purify the first protein I ever purified - a periplasmic non-specific endonuclease from an anaerobic bacterium. Had to process over 100 litres of culture by the time I was done. Although this was in the 90s, at the time we didnt know the gene sequence - the genome of the bacterium has since been sequenced.

I'm very thankful for these traditional purification experiences and that my training wasn't exclusively in the era of "overexpression of an affinity tagged protein in E. coli with subsequent one-step purification on the benchtop".

SRM said...

Also interesting to see Laemmli was one of your examiners. Was he in the room or did he participate via Skype? ;-)

SRM said...

...nevermind, I see he was at Princeton.