Recorded: 16 Jan 2003
I had been working as a protein chemist with Mike Waterfield. And we had been trying to do protein sequence on the envelope viruses, influenza virus, and I was working more on Sendi virus, on the coglycoproteins and there was some—micron (??) and a student called John had got quite a long way with flu hemagglutin and what they sequenced—what was probably they estimated about two-thirds to three-quarters of the protein but they just couldn’t get any more sequence.
And I was starting to do the same with Sendi fusion protein and the same thing, you could get some sequence—but you just couldn’t find others. And Mike suspected that these were hydrophobic regions that got—just didn’t purify through the columns that we were using.
Anyway so I went to Ashley’s seminar, which was the first sort of clear exposition of recombinant DNA techniques and so on. He was talking about their work cloning the fiber message. And it was one of those sort of light bulb moments for me where—I sort of went—you know, if it was a comic strip you’d have a cloud with a light bulb, you know, in it. And it was clear this is what we ought to be doing for the flu hemagglutin and then Sendi virus. So I went up to talk to Ashley after the seminar and he said, sure, it would be sensible but why didn’t I go and talk to Joe, and so I did. And we had wonderful discussion about it and came to the conclusion that Sendi would be too difficult. The Sendi virus genome is a single RNA of about, I think it’s five or six kilobases far outside what was possible in those times. This is the very, very early days. But the flu RNA genome is segmented with one piece for each of the proteins that are encoded and the one I was interested in was only about 1.4 kilobases so it was long for those times, but in the realms of possibility.
However, this was a negative strand bar so it didn’t have a poly-A tail. So as we discussed, we realized it would be crucial—to first of all I’d have to add a poly-A tail. And luckily Bob Kamen who was upstairs at ICRF had done a Ph.D. on poly-A polymerase, which was the enzyme. Now I believe at that time there were no commercially available enzymes or maybe one or two and certainly not poly-A polymerase. So I had to start this project by making poly-A polymerase and so Bob gave me the technique. I had to go off to Porton Down, which was the biological research establishment in England because they were probably doing quite nasty things, but you could get sort of kilogram amounts of frozen-down bacteria. So I went up there and collected a large sort of lump of E. coli bacteria and came back and went through the prep. And it went wonderfully and adding the poly-A was fine.
So I now had an RNA prep with poly-A added. Joe and Ashley by now had gone back to Cold Spring Harbor. So there was no cloning manual then, there were no techniques written up, it was all word of mouth. So I did the whole project by phone to Joe.
I mean today cloning by phone, you call and somebody sends you the clone. Then cloning by phone was you called and somebody tells you what to do next to make your own. So I would call Joe and he would say, “Right, yes, it’s first strand next. The best enzyme is, you know, so and so.” And so you call—I can’t remember [if] it was Walter Keller anyway, he would give me the phone number of somebody in some country and I would call “Hello, this is…” I was young postdoc then. “This is Mary Jane Gething. Joe Sambrook told me to call you.” And they would. Joe at that stage, the name opened all doors of course, so—and Walter Keller would send the enzyme or say the next best technique so I’d struggle away for a few weeks and finally get it to work and call Joe again. “Second strand”—no second strand was Walter Keller, I remember so call Walter Keller.
Finally I had some cDNA, at the stage you tailed the DNA which was a real pain. And again, there was a guy in the building who had the teeniest amount of terminal deoxy-transerase which was the enzyme needed. And he would dole it out to me in half microliter lots, so for every experiment I’d have to go back to him for my half microliter bit of enzyme.
And then finally I got tail DNA and tail plasmid. And called Joe because I couldn’t—using the standard transformation procedures at that time—I just couldn’t get enough clones and so I called Joe and he said “alright, you have to call Chicken Man in Harvard.” So I said, “Chicken Man?” He said, “Yes, and here’s the phone number.” So I get on the phone again. So somebody answers—I say, “Is there someone there called Chicken Man?” They said, “Oh, yes.” It turned out to be Doug Hanahan at Harvard and he was called Chicken Man cause he was current cloning chicken globin—uh, chicken globin? Anyway, chicken something. And he had just formulated liquid gold which was the famous transformation mix which he patented and as I understand has done very well at.
So he very kindly sent me a couple of flasks of this liquid gold and it really looked like there was a shimmer in it. It was a gold color with a shimmer and that worked. So I cloned the flu HA and sequenced it. Now nobody at this time would understand what sequencing was like then. It was still Maxim-Gilbert sequencing, so slow and painful. I mean, there’s a nasty step that you had to do in the fume hood and I always used to sort of shake like this while I was doing it, but by that time enzymes were coming along and New England Biolabs had started up and we would get shipments of the newest enzyme from New England Biolabs as each time they put a new one on the catalogue it would get shipped over from the States. So it was becoming easier to clone.
Then finally we had the clone and it was time to express it. I mean obviously the scientific problem we’d been working on till then was to compare different flu hemagglultins because there are different epidemics and pandemics of influenza and the viruses continually evading the immune system. But it wasn’t understood at a molecular level then why. And how the immune system—I mean how the virus evaded the immune system. But it was suspected that there were obviously mutational changes in the coat proteins and particularly the hemagglutin and so that the project was to sequence and compare different strains and I cloned in sequence two and there are other people around the world who were doing different influenza strains.
Mary-Jane Gething, biochemist is Head of the Department of Biochemistry and Molecular Biology at the University of Melbourne where she earned her Ph.D. in Biochemistry in 1974. Subsequently she went to Cambridge to do post-doctoral work.
In 1976, she moved to London to work on protein sequencing and in 1980, Gething and Joseph Sambrook received a NATO grant for travel to collaborate on virus research. She began working at Cold Spring Harbor Laboratory in 1982 where she continued her research of proteins. In 1985, Gething and Sambrook moved to Dallas to work at the University of Texas Southwestern Medical Center. They moved back to Australia in 1994.
Her current research involves protein folding in the cell and the role of molecular chaperone BiP.