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Saturday, February 04, 2012

An Ode to λ

I grew up in the phage group and spent many summers at the phage meetings in Cold Spring Harbor. Back then (late 1960s, early 1970s), the best scientists worked on bacteriophage λ (lambda) and the rest of us just tried to keep up.

A number of key insights in molecular biology came from studying this small virus that infects Escherichia coli and if you didn't know about that research you were really out of the loop.

But by 1990 it was already apparent that a new generation of students was growing up in ignorance of the fundamental concepts learned from studying bacteriophage and bacteria. I remember asking a class what they knew about the genetic switch in bacteriophage λ and getting nothing but blank looks! Everyone worked on eukaryotes by then and the knowledge acquired by the phage group was not relevant.

I tried to teach that knowledge in my classes. In my textbook I devoted 27 pages to describing the regulation of phage genes (in a chapter on "Gene Expression and Development"). Other instructors didn't care.

Here's a short list of things we learned from studying λ. How many have you learned?

  1. Mutants in the E gene of λ can be rescued by an important class of bacterial mutations in two different genes: groE and groL. What do the products of those genes do?
  2. The integration of λ DNA into the bacterial genome to form a prophage occurs at a single specific site in the bacterial chromosome. How does this classic example of site specific recombination work?
  3. The λ genome is a linear molecule 49kb in length. All of it is replicated accurately, including the ends. How does this work?
  4. The phage has dozens of genes but only two of them are transcribed immediately after infection (immediate early genes: N and cro). Why aren't the others transcribed?
  5. The product of gene N is an antiterminator and it's expression allows transcription of several early genes (cro, cII, O, P, Q, cIII, Xis, and Int). How does antitermination work and what did we learn about transcription complexes and the importance of NusA?
  6. Very early in a lytic infection there is a small RNA (6S RNA) produced in huge amounts from the PR' promoter. The RNA does not encode any protein and has no function. Why is it produced?
  7. cII is a well-studied example of a transcriptional activator that recognizes multiple promoters. How does it work?
  8. The λ repressor (cI) is both a repressor and an activator. So is cII. We now know that many transcriptional regulators function as both repressors and activators. What is the mechanism?
  9. Two transcriptional regulators, cI and cro, bind to the same six operators but have opposite effects. We now know of many examples of this kind of competition. How does it work?
  10. When sufficient copies of lambda repressor (cI) are made, transcription of all early and late genes is repressed. Why doesn't this happen during a normal lytic infection?
  11. In order to induce the prophage, the repression by cI has to be blocked. What circumstances cause this and what is the mechanism?
  12. The expression of the Q gene is inhibited by an antisense RNA. How does antisense RNA regulate gene expression?
  13. The structures of cI and cro revealed a universal DNA binding domain found in many transcriptional regulators, including the HOX proteins. What is that domain and how does it interact with DNA?
  14. One of the strong leftward promoters, PINT, is right in the middle of the xis gene and this is related to control of integrase synthesis by regulating the stability of its mRNA. How?
  15. The genetic switch is an excellent and very well understood example of developmental regulation of gene expression. Do you know how it works?
  16. Bacteriophage λ infection is enhanced by maltose. This led to an understanding of cell surface receptors. How?

[Image Credit: The bottom two figures are from Microbiology 2nd ed. by J.L. Slonczewski and J.W. Foster, W.W. Norton & Co. Inc., 2010]


Anonymous said...

I think lambda is still discussed in most genetic classes and 10 years ago I went into it in depth in a graduate class. It seems to be that it should always be discussed because of the elegance of the experiments that disected the circuits...but I'm not sure how relevant it is for eukaryotic gene expression...and thats why they begun the research- to get a handle on eukaryotic expression ( not that knowledge of other organisms isnt usefull for its own sake)

Rod W

DK said...

As an undergrad, I would have been able to tell basics of the switch and life cycle and that's all (the knowledge would have come from molecular genetics class, not biochem). Today I know a little more but still I can answer only five of the above adequately and can mumble something semi-meaningful on three more.

Psi Wavefunction said...

Yeah, ok, I shamefully missed out on basically all of this, despite majoring in a cell/molbiol stream in my undergrad. You see, this would've fallen under MICRO courses, which were highly competitive and very medically oriented (we had not a single course on real bacterial diversity, for example). I think this is a big problem with the shift in undergrad biol curricula from fundamentals of biology towards applied pre-med type stuff, especially when this happens at the level of the whole program, ignoring the 5%* of us who are actually not pre-meds. On a related note, saddening how most older folk (and those from abroad) are at least somewhat aware of the existence of protists and are familiar with some of their key contributions to biology. Most modern American and Canadian biol graduates, incl grad students, hardly know what 'protist' is, and would be shocked to discover that that obscure kingdom is host to many crucial discoveries like telomerases in ciliates and much of the fundamental actin-myosin work in Amoeba+Chaos, for example.

I don't even know if it's overspecialisation because it's not like you graduate a BSc program with any particular expertise in anything...

I'll go read up on phages now.

*made-up statistic

Anonymous said...

In Cologne, I had lectures with Benno Müller-Hill, the guy who, together with Walter Gilbert, isolated Lac-Repressor, first.
Benno tried hard to interest us in these topics (that was around 1995). And he succeded (in part).
I would estimate that some 10% of my year actually read "A genetic switch"...
And that was due to his effort. In retrospective I do not regret it.

By the way, one reason for me to read 'Sandwalk' is that I (as a PhD holding biologist)learn here a lot about basic biochemistry (not to talk about central dogmas and random genetic drift).

Thanks a lot for the blog!

gillt said...

Incidentally, lambda is the sign I use instead of ul (microliter). I have no idea of the precedent, but that's how I was taught. Plus it takes up less space on tubes.

SPARC said...

Müller Hill's "The lac operon" is a good addition to Ptashne's story on lambda's genetic switch.
It is still available:
Unfortunately, not too many students using blue/white screening are aware of where alpha complementation derived from.

student said...

I actually took a 4th year CSB class at U of T last semester where we discussed the lambda phage switch for the first 2-3 weeks and were assigned most of Ptashne's book. One of the best courses I've taken, all about regulatory networks.

Anonymous said...

Some basics of the switch are now taught in B10130 and BIO230 at U of T; and the GroE/GroL pathway is mentioned in passing in BCH340 as a chaperone mechanism.