The Early Days of Restriction Enzymes
Recently, I met up with John Collins who, in collaboration with Barbara Hohn, invented cosmids in 1978. We took delight in reminiscing about the early days of recombinant DNA. A few days after our meeting, John offered this delightful telling of the story of one particular restriction enzyme. —Roberto
by John Collins
In the early 1960s, Werner Arber in Switzerland discovered that foreign DNA entering a bacterial cell could be cleaved on both strands by restriction endonucleases (restriction enzymes). This was part of the host's defense against viral attack. For this discovery he shared the 1978 Nobel Prize with Hamilton Smith and Daniel Nathans. Smith and Nathans had in turn discovered restriction enzymes from Haemophilus influenzae and shown by Smith to recognize specific sequences and cleave SV40 viral DNA into many specific fragments that could be separated and visualized (if labelled with 32P) using polyacrylamide gel electrophoresis. They predicted that these enzymes would be useful for generating small DNA fragments for DNA sequencing. Not only did these unique enzymes greatly facilitate sequencing, but they also helped usher in the era of DNA cloning – initiated by Herb Boyer, Stan Cohen and colleagues in 1973 – and aided in the discovery of RNA splicing in 1977, through the independent work of Phil Sharp and Rich Roberts and their teams (Nobel Prize 1993).
Roberts realized the enormous potential of restriction enzymes and engaged many of the scientific community to search for more. Consequently, during the 1970s many scientists were discovering and purifying many new restriction enzymes from diverse bacteria in the labs and swapping or donating aliquots of these with colleagues at other institutes. We at the National Center for Biotechnological Research in Braunschweig, Germany, routinely made SmaI from Serratia marcescens, and EcoRI (from Herb Boyer's Escherichia coli strain). In 1974 Donald Comb founded New England Biolabs (NEB) with close connections to several scientists. The declared intention was to supply research materials and then plough some of the profits back into academia. Rich Roberts became involved in NEB as Chief Scientific Officer. For taking on this post, Roberts initially got a lot of criticism since "entrepreneur" was a dirty word for life scientists in those days, tainting his or her reputation. I had more than my share of that when my institute wanted to patent the cosmid-cloning technique 1977.
Fig. 1. Edge of a living colony of Caryophanon latum incubated for 24 hr. at 26°C. The filaments are closely subdivided by numerous cross-walls. The two refractile biconcave segments in the central filament are disintegrated cells, × 1700. Source
An amusing anecdote from this time occurred to a colleague of mine. Hubert Mayer, who was teaching a biology course at the local University in Braunschweig. He initiated a search for restriction enzymes in novel organisms. One of the organisms was Caryophanum latum, a very large bacterium isolated from cow-dung that inevitably lysed when it reached stationary phase in laboratory culture. To Hubert's amazement, the crude supernatants of the lysed cultures contained no non-specific nucleases. Rather, these supernatants contained a single restriction endonuclease, which by convention was called ClaI. This enzyme's cleavage pattern of lambda DNA clearly implied it had a completely new specificity. Indeed, ClaI cut the widely used cloning vector pBR322 at a single site between the tetracycline resistance promoter and the structural gene, making it very useful for cloning. We offered NEB the strain for their commercial use asking for a couple of thousand German marks (about US$700 at the time) to go back into our research fund. NEB said they didn't want to pay for the strain. And yet, they asked us for all the details as to how to prepare the growth media and everything related to cultivating this fastidious bacterium. Turns out, the simplest-to-make and most often used medium was prepared from the bacterium's natural habitat. That is why Hubert brought cow-dung to the lab every morning to prepare the growth medium! We informed NEB of the medium composition and our way of preparing it. They apparently thought we were joking and trying to take the Mickey out of them. Writing back (people wrote letters in those days!), NEB replied "…if we give anyone any money, it will be the farmers." They took our C. latum strain anyway, never giving us any funds. Yet, I am sure they profited; in 2015, NEB was valued at US$ 1.6 billion.
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John Collins (born 1945) pioneer in gene technology, co-founder of HUGO; introduced gene technology to Denmark and Germany (1974/5); retired Professor at University of Braunschweig and the GBF (now HZI) Braunschweig; since 2012 science historian. Self portrait: a curmudgeon brimming with untold tales of academic, civil servant and industrial hypocrits.
