Well, I was pretty sure there was some sort of connection between MPF and cyclin. MPF was still just an activity at this stage. No one had any kind of molecular handle on it, and I didn’t dare even to hope there could be a direct connection. There were two problems with cyclin actually being MPF as I saw it at the time. One was that entry into mitosis is a rather abrupt, switch-like business, whereas accumulation of new protein is a rather linear business, so how do you turn a gradual increase in something into a sharp, switch-like response? The other thing that bothered me was the question of whether you could really make enough new enzyme in 15–20 minutes that it could catalyse a cell cycle transition? That seemed to me a bit unlikely. So we preferred the rather vague idea that cyclin might be a hypothetical anti-anti-MPF.
It took ages and ages to figure out what was really going on. Of course we now know that there is an elaborate regulatory machinery involving phosphorylation that helps to account for the switch-like response — but at that time we really had no information about what we were dealing with. We needed to clone and sequence the wretched thing. Luckily, there was absolutely no interest, and the cyclin paper was hardly cited at all for the next 5 years. Probably ‘wild speculation based on faulty logic’ was many people’s reaction. I was getting very sceptical looks from people and my pals in Cambridge: ‘Tim’s gone off his rocker’. Everybody says they now remember me showing them this gel and how excited I was, but obviously I wasn’t very good at explaining why. I wasn’t too good at articulating it, because really there wasn’t anything to articulate. I just knew it was right, but the details were totally unknown and unknowable actually at that stage.
The next great advance came in the summer of 1986 at Berkeley, California. By that time, we knew there was more than one cyclin, though we didn’t know why, and Eric Rosenthal had cloned cyclin A from clams when he was a graduate student with my friend Joan Ruderman. Joan and her postdoc Katharine Swenson had done the brilliant experiment of injecting cyclin messenger RNA into frog eggs, which then matured [4]. That really put the cat among the pigeons. I wanted to repeat that for a start. We did it using RNA from Urechis eggs, a degenerate annelid worm from the mudflats of northern California that Eric was working on — and that worked, and then we wondered whether, if we made mRNA from mature frog eggs, whether that would work — and it did. John Gerhart’s technician Mike Wu had never ever seen an oocyte mature in response to injected messenger RNA, and he’d injected absolutely everything — he was the ace injector in the Bay Area — so we were tremendously excited. We went back to the lab at ten o’clock at night, and there the white spots were forming (Fig. 4)…it was such a simple experiment! So frog eggs contained mRNA that made frog eggs mature.
Then you really knew. Previously cyclin was a protein that came in weird organisms like sea urchins and clams, and nobody cares about sea urchins or clams, and then Urechis, and that’s even more obscure. But to have frog mRNA catalyse its own maturation. Wow! Tada! We were home and dry and knew we’d be able to find the cyclin mRNA in frogs. Jon Pines had been working with me on cloning cyclin from sea urchin eggs, and once he had a clone for sea urchin cyclin B, we screened frog cDNA libraries, and sure enough there were positives and we sequenced them and sure enough they all had the same or related sequences. We didn’t go after human cyclin because Jon graduated and went off to work with Tony Hunter with the idea of looking for the human cyclins. It was very quick and easy for him then to pick up that ball and run with it, which was very gratifying.