Wednesday, November 29, 2006

Phage Renaissance

Bacteriophage, or phage, occupy an exalted place in the history of modern biology. Hershey & Chase used phage to nail down DNA (and not protein) as the genetic material. Benzer pushed genetic mapping to the nucleotide level. And much, much more. Phage could be made in huge numbers, to scan for rare events. Great stuff, and even better that so many of the classic papers are freely available online!

Phage have also been great toolkits for molecular biology. First, various enzymes were purified, many still in use today. Later, whole phage machinery were borrowed to move DNA segments around.

Two of the best studied phage are T7 and lambda. Both have a lot of great history, and both have recently undergone very interesting makeovers.

T7 is a lytic phage; after infection it simply starts multiplying and soon lyses (breaks open) its host. T7 provided an interesting early computational conumdrum, one which I believe is still unsolved. Tom Schneider has an elegant theory about information and molecular biology, which can be summarized as locational codes contain only as much information as they need to be located uniquely in a genome, no more, no less. Testing on a number of promoters suggested the theory valid. However, a sore thumb stuck out: T7 promoters contain far more information than the theory called for, and a clever early artificial evolution approach showed that this information really wasn't needed by T7 RNA polymerase. So why is there more conservation than 'necessary'? It's still a mystery.

Phage lambda follows a very different lifestyle. After infection, most times it goes under deep cover, embedding itself at a single location in its E.coli host's genome, a state called lysogeny. But when the going gets tight, the phage get going and go through a lytic phase much like that of T7. The molecular circuitry responsible for this bistable system was one of the first complex genetic systems elucidated in detail. Mark Ptashne's book on this, A Genetic Switch, should be part of the Western canon -- if you haven't read it, go do so! (Amazon link)

With classical molecular biology techniques, only either modest tinkering or wholesale vandalism were the only really practical ways to play with a phage genome. You could rewrite a little or delete a lot. Despite that, it is possible to do a lot with these approaches. In today's PNAS preprint section (alas, you'll need a subscription to get beyond the abstract) is a paper which re-engineers the classic lambda switch machinery. The two key repressors, CI and Cro, are replaced with two other well-studied repressors whose activity can be controlled chemically, LacI and TetR. Appropriate operator sites for these repressors were installed in the correct places. In theory, the new circuit should perform the same lytic-lysogeny switch as lambdaphage 1.0, except now under the control of tetracycline (TetR, replacing CI) and lactose (LacI, replacing Cro). Of course, things don't always turn out as planned.
These variants grew lytically and formed stable lysogens. Lysogens underwent prophage induction upon addition of a ligand that weakens binding by the Tet repressor. Strikingly, however, addition of a ligand that weakens binding by Lac repressor also induced lysogens. This finding indicates that Lac repressor was present in the lysogens and was necessary for stable lysogeny. Therefore, these isolates had an altered wiring diagram from that of lambda.
. When theory fails to predict, new science lies ahead!

Even better, with the advent of cheap synthesis of short DNA fragments ("oligos") and new methods of putting those together, the possibility of becoming the "all the phage that's fit to print" is really here. This new field of "synthetic biology" offers all sorts of new experimental options, and of course a new set of potential misuses. Disclosure: my next posting might be with one such company.

Such rewrites are starting to show up. Last year one team reported rewriting T7. Why rewrite? A key challenge in trying to dissect the functions of viral genes is that many viral genes overlap. Such genetic compression is common in small genomes, and gets more impressive the smaller the genome. But, if tinkering with one gene also tweaks one or more of its neighbors, interpreting the results becomes very hard. So by rewriting the whole genome to eliminate overlaps, cleaner functional analysis should be possible.

With genome editing becoming a reality, perhaps it's time to start writing a genetic version of Strunk & White :-)

1 comment:

Ping said...

Here is a mathematical solution
to the phage lambda stability puzzle, with a few predictions:
1) Calculating Robustness of Epigenetic States in Phage lambda Life Cycle,
X.-M. Zhu, L. Yin, L. Hood, and P. Ao, Functional and Integrative Genomics 4: 188-195 (2004).
2) Robustness, Stability and Efficiency of Phage lambda Regulatory Network: Dynamical Structure Analysis,
X.-M. Zhu, L. Yin, L. Hood, and P. Ao, Journal of Bioinformatics and Computational Biology 2: 885-817 (2004).
3) Efficiency, robustness and stochasticity of gene regulatory networks in systems biology: lambda switch as a working example.
X.-M. Zhu, L. Yin, L. Hood, D. Galas, and P. Ao, Chapter 18, in Introduction to Systems Biology, (Humana Press, 2007, in press):
(arXiv: q-bio/0512007. )

The results are so quantitative and the agreement with experimental data appears so good (from a bioloigcal point of view) sometimes even I could not believe
what we have achieved.