Christoph's recent post on visualizing introns brought back wonderful memories. In mid-1976, I spent the first several months of my PhD visualizing replicating plasmid DNA. I loved it! Here's why. Molecular biology developed largely during the 1940s, 1950s and 1960s. By then genetics and biochemistry yielded a good picture of how transcription and translation occurred and how they were regulated.
DNA replication was a bit different. Though it was apparent from the double helix model how replication could proceed, and later proven by Meselson and Stahl (the most beautiful experiment), a clear understanding of the genetics, biochemistry and regulation of DNA replication lagged. Nonetheless, by the 1960s several lines of microscopic evidence indicated that in bacteria DNA molecules were often circles (see for example, STC Pictures Considered #2) and that replication appeared to start from unique sites (origins of replication). In 1963, Jacob, Brenner and Cuzin proposed their "replicon" hypothesis: an "initiator" (protein or nucleic acid) would act at an origin to regulate initiation events. Many investigators were inspired by this proposal and became interested in studying the regulation of replication. Plasmids, because of their dispensability and small size seemed ideal model replicons to determine if the replicon hypothesis was correct. Direct evidence supporting it would come, but not for some fifteen years.
At the outset, one of the key tools available to study replication was DNA visualization using electron microscopy. This technique was published in a 1959 paper by Albrecht Kleinschmidt et al., and afterwards was widely known as "Kleinschmidt spreading." Throughout the 1960s and early 1970s investigators used it to "see" DNA replication. A hugely important addition to this analysis was to use it in combination with restriction enzymes, allowing the mapping of origins relative to where restriction enzymes cut the DNA (e.g. Fig. 2). Origins could be localized! What great excitement that was! I hope you will understand why, in 1976, I was so enthralled spending endless hours analyzing plasmid replicative intermediates using electron microscopy.
OK, we could visualize origins... But what were these origins? Were there really initiators acting on them? Clearly Kleinschmidt spreading alone would not suffice to figure this out. Many of us turned to using restriction enzymes differently. We used them to define origins functionally, i.e., we cloned them. That meant abandoning my beloved electron microscopy, replacing it with cloning and, quickly afterwards, DNA sequencing. In a matter of months, many of us went from simply seeing origins by Kleinschmidt spreading to defining their size, their sequence, their functionality and identifying initiators that acted on them (for my personal example, see here). There it was, direct evidence supporting the replicon hypothesis! The field had taken a quantum leap.
There's a take-home message to this story that still applies today. Powerful methodologies come and go. My recommendation is that experimentalists should always be ready to abandon their most beloved techniques as soon as they perceive that they have reached their limits. There might be better approaches to address the questions under investigation. If you are an experimentalist, you should constantly be on the lookout for (or developing yourself) new tools that might lead you to deeper insights. And don't wait for these tools to be handed to you in the form of a ready-made kit!
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