Thread by @somssichm

Do you know who Douglas Prasher is? He is the person who cloned the original GFP gene in the early 1990s. In my short history of plant light microscopy I also cover a bit of his story - & why he is relatively unknown today, despite the importance of his work. See here: 1/14 🧵

https://twitter.com/somssichm/status/1386558825088552962

2/14 In 1962 Osamu Shimomura et al. identified the bioluminescent Aequorin in the Aequorea jellyfish, as well as a green fluorescent protein, that seemed to act as a FRET-acceptor for the Aequorin 'in jelly' [1][2][3] (REFs at the end).

3/14 20 years later, Milton Cormier aimed at cloning the Aequorin gene to use it as a bioluminescent marker for use in diagnostics. He hired Douglas Prasher for this job. [4] In 1985, Prasher et al. published the successful cloning, expression and in vitro function Aequorin. [5]

4/14 Prasher, however, felt that the GFP was a much more interesting gene to use as potential reporter, as it seemed to be fluorescent without any co-factors. So he took up a tenure-track position Woods Hole Oceanographic Institution with the aim to clone the GFP gene.

5/14 His time at Woods Hole did not go as planned, however. He felt unsupported and isolated as a molecular biologist at an Institute made up of marine biologist, and nobody seemed to share his vision that GFP could function as a stand-alone fluorescent reporter.

6/14 When another of his grant applications was turned down by the NIH, Prasher had grown so frustrated and depressed, that he decided to stop his tenure-track process and quit his job following the successful cloning and publishing of GFP in 1992. [6]

7/14 By coincidence, two scientists who did share Prasher's vision, both searched the Medline database for information on GFP in early 1992, right when Prasher's paper was published. One was Martin Chalfie, who had actually contacted Prasher previously concerning GFP. [7]

8/14 The second was Roger Tsien [8]. Upon finding his paper, they both immediately asked Prasher to share his GFP clone. Sending out the GFP clone to Chalfie and Tsien turned out to be the last act of Prasher, before he left Woods Hole and his academic career behind.

9/14 Upon receiving the GFP clone from Prasher, things went fast for Chalfie and Tsien. Chalfie demonstrated its usefulness as a protein tag in E. coli and C. elegans, & then passed the clone on to his wife, Tulle Hazelrigg, who demonstrated its usefulness in Drosophila. [9][10]

10/14 Chalfie had already included the info on GFP's functionality in Drosophila in his paper, however, for which he made sure to get Hazelrigg's written permission beforehand. Permission was granted subject to three specific conditions (see letter in image). [7]

11/14 Roger Tsien, on the other hand, used the GFP clone as a starting point to design a whole suit of new fluorophores of all colors and with different features, starting with an improved GFP, a BFP, CFP and YFP, just between 1994 and 1996. [11]

12/14 A view of the GFP 'family tree' on FPbase.org gives a good impression of the work Tsien and his successors have done - based on Douglas Prasher's clone. [12]

13/14 In 2008, Chalfie & Tsien, together with Shimomura, received the Nobel Prize in Chemistry for their work on ‘the discovery & development of the green fluorescent protein, GFP'. [7][8][3] At the time, Douglas Prasher was working as a courtesy van driver for a car dealership.

14/14 Thanks to an invitation and the support of Tsien and Chalfie, Prasher and his wife were able to attend the Nobel Prize award ceremony, and his essential contribution was always acknowledged by all three Nobel laureates. [7][8][3] And yet, the prize itself eluded him.

REFs: [1]doi.org/10.1002/jcp.10… [2]doi.org/10.1002/jcp.10… [3]doi.org/10.1002/anie.2… [4]discovermagazine.com/mind/how-bad-l… [5]doi.org/10.1016/0006-2… [6]doi.org/10.1016/0378-1… [7]doi.org/10.1002/anie.2… [8]doi.org/10.1002/anie.2… [9]doi.org/10.1126/scienc… [10]doi.org/10.1038/369400…

[11]doi.org/10.1146/annure… [12]fpbase.org/protein/avgfp/

This and more in my 'A short history of plant light microscopy': zenodo.org/record/4719063