Thursday, March 26, 2009

Thesis Defense - 35th Anniversary

 
Today is the 35th anniversary of my Ph.D. oral defense. 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.")


16 comments :

  1. Congrats on 35 fruitful years. Proud to have been your student for one of them.

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  2. I thought this post might get into the "Old Tools" category! :) Just kidding. How well I remember that damn thesis. In those days, we had no computers and every friggin' time you made one small revision (count them) the entire chapter had to be retyped. Who said that taking typing in high school was a dumb option?? Turns out I was responsible not only for getting you out of Princeton, but also our lawyer! It was such a wonderful time in our lives. It was always the goal to leave, and carry on with new research, but how I wish we were able to relive those days now. Congratulations and best love always.
    Leslie Jane.....

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  3. 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.

    Early evidence for Intelligent Design!

    Congratulations, Larry and Leslie Jane.

    Truti

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  4. Jeez, I can't even remember the date of my defence. It never felt particularly important. Just another formality...

    So, you learned a lot as a graduate student but you haven't answered the scientific question about gp41. Sh#t happens. Then, what did you do as a postdoc (and for how long)? And, what did your publication record look like when you got a real job (=tenure track)? And after that, what were important findings that earned you a tenure?

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  5. Congratulations! Even though I have no idea what you're talking about, it sounds important, and surely earning your PhD is a huge milestone. Love the photo!

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  6. Aside from the haircut, you looked far more innocent back then. Congratulations on the anniversary of your achievement!

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  7. I have heard stories about this 'typewriter' thing. And the 'hand drawn figures'... I thought they were just scary stories PIs told their students to give them nightmares, like The Boogie Man.

    I dont know how you all did this without computers. You all are amazing.

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  8. DK asks,

    Then, what did you do as a postdoc (and for how long)?

    I spend four years in Geneva Switzerland with Alfred Tissières studying heat shock genes in Drosophila melanogaster.

    And, what did your publication record look like when you got a real job (=tenure track)?

    Pretty decent. I had four papers from my work as a graduate student and seven from my postdoc days (three were in press). All of the postdoc papers were collaborations with Walter Gehring's group in Basel, Switzerland.

    And after that, what were important findings that earned you a tenure?

    There weren't any. I convinced them to give me tenure based on future promise. I think they regret the decision. :-)

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  9. I find your story inspiring. I am going through a similar event now as I try to wrap up my PhD and it would not be possible without the love and support of my wife.

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  10. I convinced them to give me tenure based on future promise. I think they regret the decision. :-)

    So you lucked out. Good. Judging from the blog, you are probably a very good teacher. That alone, IMHO, should qualify for a tenure. But, alas, that's not how it really works - particularly these days.

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  11. DK says,

    So you lucked out. Good. Judging from the blog, you are probably a very good teacher. That alone, IMHO, should qualify for a tenure. But, alas, that's not how it really works - particularly these days.

    I'm not sure how to interpret your comments.

    If you have a point, could you make it more clearly?

    The rules for tenure in my department haven't changed very much in the past 30 years. Research productivity and promise are the only things that count. In my department the decision is made in your fourth year as an Assistant Professor.

    Do you think the rules have changed?

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  12. I don't really know how things were 30 years ago so I can't be sure how things changed. But the trend in tenure, I think, is to limit it. See, for example:
    http://journals.lww.com/academicmedicine/Fulltext/2004/03000/Tenure_in_Transition__Trends_in_Basic_Science.3.aspx

    What I do know is that in decent research universities these days getting a tenure based on future promise (like you say you got) is pretty much impossible. One must have a major grant and a very good to outstanding publication record. Assuming that you were hired in 1978-79 and got tenured in 1984, you had four papers published by that time. Two of them in major journals (PNAS and MCB) and two in obscure ones (Can J Biochem Cell Biol and Bioscience Rep). In places I know, that would never be enough. In fact, a person in my dept. was recently denied tenure despite having a five years NSF grant and 9 publications in major journals an Assistant Prof.

    So one of these:
    1. You simply got lucky.
    2. Standards today are more stringent.
    3. U Toronto's standards are nowhere near standards in the ~ top 50 US universities.

    Personally, I think it's a combination of 1 and 2.

    That said, I do feel you deserved tenure if you are a very good teacher. Screw publication records and funding. Quality of education is in steady decline precisely because
    of the emphasis on faculty bringing in extramural megabucks. I am sick and tired of professors who view teachng as a major distraction from their real job (getting funded to do research).

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  13. DK,

    I have no intention of debating you about whether I qualified for tenure 25 years ago.

    If you have a point then please make it more clearly. Are you whining about the fact that things seem so much tougher for you than they were for me? Boo hoo.

    Does it make you feel better to belittle my achievements?

    Different universities have different ways of making decisions and who's to say which one is correct? The kind of effort it took to publish a paper in the past isn't the same as it is today.

    To me it sounds insane to deny tenure to the person you mentioned. However, I know there are some universities that have a very high opinion of themselves.

    Care to mention which place you're talking about?

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  14. I have no intention of debating you about whether I qualified for tenure 25 years ago.

    If you have a point then please make it more clearly. Are you whining about the fact that things seem so much tougher for you than they were for me? Boo hoo.


    Sorry, I can't make my point more explicit than I already have: I think you were qualified for a tenure because, as far as I can tell, you are a good teacher. Still, either you lucked out or things are much tougher today than 25 years ago. So, what is it? Care to answer?

    And no, I don't think that merely stating the facts constitutes whining.

    To me it sounds insane to deny tenure to the person you mentioned ...
    Care to mention which place you're talking about?


    No, I am not naming names. Because this is not about myself. If you allow anonymous responses, you shouldn't have a problem with it.

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  15. Great post! And congratulations post factum!

    In that photograph you look a bit like my dad! (*1943, MD 1972/73, I think.) Obviously, there has been some divergence since then (except in the hair department, for all I can judge).

    And yes, I do hear stories about typewriters, and punchcards, and all that stuff, sometimes. My mom was not involved in the diss, AFAIK. Gotta ask, though.

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  16. 35 years of Thesis Defense is a very long period!

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