Pictures Considered #2. The E. coli Chromosome Caught in the Act of Replicating

By Elio

A radioautograph of the E. coli chromosome in the act of bidirectional replication. The image shows a circular molecule with two “rabbit ears”, representing the newly replicated regions. E. coli DNA was labeled with tritium-labeled thymidine by growing the cells for two generations in the presence of this specific DNA precursor. Cairns had developed a method for depositing intact DNA molecules on a microscope slide, then covering it with a photographic emulsion. The slide was then stored in dark for two months to allow the tritium to decay and sensitize enough photographic grains to reveal patterns after photographic development, as seen in this radioautograph. The insert on the right corner of the image shows a tracing of the replicating chromosome, which is a circle nearly 1 mm long. From Cairns, J.P.: Cold Spring Harbor Symposia on Quantitative Biology 28:44, 1963.

We continue our series of images that have made a difference in microbiology. This one, published by John Cairns in 1963 has earned a most deserved place in textbooks and reviews. It shows a radioautograph of an intact E. coli chromosome in the act of replicating. From this image, Cairns concluded that the E. coli chromosome is:

• a single piece of double stranded DNA, 700-900 μm long, and
• circular.

These truths had been surmised from other, mainly indirect pieces of evidence. For instance, the idea that the genome consists of a single chromosome had already been inferred from the fact that only a single genetic linkage group could be found in E. coli. Its circular nature had been suspected because all the genes on the chromosome were circularly permuted. But this image gave these notions physical authenticity, ergo finality. Such important conclusions from one single picture!

In addition, by labeling the newly replicated DNA in the chromosome with a short pulse of tritiated thymidine, Cairns provided conclusive evidence for the bidirectionality of chromosome replication, with each of the two replicating forks containing one new and one old DNA strand.

Correction

As pointed out by Christoph Weigel and David Lane, I wrongly attributed to Cairns the evidence that the E. coli chromosome replicates bidirectionally. In fact, the definitive proof stems from a later elegant radioautographic study by Prescott and Kuempel and several equally convincing genetic experiments, e.g., by Masters and Broda. A regrettable memory lapse…..

Two grain tracks produced by E. coli chromosomes from cells labeled for 13 min with [³H]thymine, followed by labeling for 2.5 minutes with [³H]thymine and [³H]thymidine.  The numbers indicate the length of the grain tracks. Each track is produced by two daughter (sister) DNA duplexes. Source.

Comments

[David Lane]

Elio - and your correspondents - you are right in saying this should be taught. It is an excellent example of how pre-conceptions lead to faulty conclusions. The molecule and its circularity are beyond dispute of course, but Cairns concluded on the basis of grain density that it was replicating unidirectionally from point X. A moment's contemplation of the interpretative diagram shows that it misrepresents the radioautogram and makes no sense in terms of replication either. In fact the molecule shown was from cells labelled uniformly (two generations, if memory serves); the real demonstration of bidirectionality was the Prescott & Kuempel paper, and at the same time Masters & Broda who used a different cunning technique.

[elio schaechter]

You mention in the last paragraph of your piece on the Cairns autoradiogram the proof for bidirectional replication. The autoradiograms shown in the Prescott & Kuempel (1972) paper are not as iconic but if you-by chance-had this picture in mind when stating "In addition, by labeling the newly replicated DNA in the chromosome..." please post the reference.

Christoph Weigel

Elio replies:

Yes, I had that in mind. The reference is: Prescott DM and Kuempel PL. Bidirectional replication of the chromosome of Escherichia coli. PNAS 69:2842-45, 1972. At: http://www.pnas.org/content/69/10/2842.full.pdf

[Bob Blasdel]

I wonder if anyone has made a picture that clear but with multiple sets of replication forks coming off of the new origins.

Using pulses of tritiated thymidine is such a powerful and straightforward technique that can produce such elegant data, its amazing that so few people use it anymore.

Elio replies,
For an old timer like myself, this is but one of the elegant techniques that were used in the past and that are nearly forgotten. I'm tempted to keep a list. So thanks for bringing this up.

[Mark O. Martin]

Oh, and the best part? After students "get" it, you get to remind them of the dangers of "colicentricity": that not all bacteria have a single, circular chromosome! Variation rules!

Elio replies to both Mark's comments.

You made toe excellent points. I'm glad someone (you) is carrying the banner.

[Mark O. Martin]

You know, Elio, this photograph isn't even taught any more in freshman biology. And it should be, because Cairns' imagery really shows how replication forks proceed in a clear and visual way. Oh well. At least---thanks to Small Things Considered---my students will see it!