Making the First Recombinant DNA Molecule

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00:00:11.14 I'm Paul Berg.
00:00:12.23 I'm a retired professor at Stanford Medical Center.
00:00:17.02 And I'm going to reminisce a little bit
00:00:19.16 about how the recombinant DNA idea emerged.
00:00:25.09 I had been carrying out experiments
00:00:27.20 with a colleague at Stanford, Charles Yanofsky,
00:00:31.17 and as part of the experiments we were doing
00:00:33.21 we were using phage-mediated transduction,
00:00:36.22 the delivery of genes from one strain into another
00:00:41.00 using a virus as the carrier
00:00:43.21 of that new genetic information,
00:00:47.21 a very important technology
00:00:50.03 that had been widely used,
00:00:51.25 and very important in the development of our current notions
00:00:54.07 of the molecular biology of microbes.
00:00:58.15 But, at the same time,
00:01:00.21 I had decided to move from my work in bacteria
00:01:03.22 to try to learn something about mammalian cells.
00:01:07.14 It was largely to try and test
00:01:09.15 whether the ideas that had prevailed
00:01:12.02 about gene function in microbes
00:01:14.22 was also applicable to mammalian cells.
00:01:17.01 And I took a year off and went to work
00:01:19.23 at the Salk Institute with Renato Dulbecco,
00:01:22.13 and to work on the tumor viruses.
00:01:24.23 And I chose the tumor viruses
00:01:27.01 because they were known to have very small genomes,
00:01:29.06 and they were known to transform normal cells
00:01:31.18 into cancer cells.
00:01:33.29 And that seemed like a nice place to start
00:01:36.28 because, again, using viruses
00:01:40.09 that have limited genetic information,
00:01:41.25 being able to follow the expression of their genes,
00:01:44.25 seemed more tempting than trying to study
00:01:48.01 the whole mammalian cell genome.
00:01:52.29 During the time I was down there,
00:01:56.00 it was reported that
00:01:58.23 when SV40, or polyoma, infects mammalian cells,
00:02:02.28 virus are produced,
00:02:05.04 but amongst the progeny there are virions
00:02:08.04 which contain only cellular DNA, and not viral DNA.
00:02:13.03 And that was very similar
00:02:15.09 to the bacterial transduction system P1,
00:02:19.03 which infects E. coli
00:02:21.09 and comes out as P1 particles,
00:02:23.05 but containing E. coli segments of DNA,
00:02:25.17 and it seemed to me interesting to think about the idea of
00:02:29.25 trying to develop a transduction system
00:02:32.19 for mammalian cells to facilitate
00:02:34.27 the genetic modifications of cells,
00:02:36.20 much as we had done with microbes.
00:02:39.03 But very quickly it became clear
00:02:41.15 that a single virus particle
00:02:43.22 could not contain very much DNA,
00:02:45.19 in fact the limit is about five kilobases.
00:02:48.14 And the prospect of being able to find a gene
00:02:51.23 that was present in the human genome
00:02:54.20 of three billion basepairs,
00:02:57.01 and find it in a virus particle
00:02:59.15 that contained five [kilobases],
00:03:01.19 seemed pretty small.
00:03:03.15 So... but I liked the idea that we could actually
00:03:05.26 introduce new genes into mammalian cells,
00:03:08.09 and we could do it without a virus particle.
00:03:10.14 We could actually take the SV40 DNA,
00:03:13.00 which was known to integrate into the cell's DNA that it infects,
00:03:17.18 and link up to it some foreign DNA,
00:03:21.12 anything we wanted to put into the mammalian cell.
00:03:24.00 And so the first idea was:
00:03:27.02 could we get two different DNAs,
00:03:29.07 join them together covalently,
00:03:31.12 and then use them as a way...
00:03:34.10 as a transducing agent?
00:03:36.07 And we had in the lab a plasmid,
00:03:40.13 lambdadvgal.
00:03:42.04 It was a small DNA molecule
00:03:44.08 about ten kb,
00:03:46.07 which had lambda genes
00:03:48.16 and appropriate genes that it could replicate in E. coli,
00:03:51.05 and linked to it were the three bacterial genes
00:03:54.00 that encode the gal operon...
00:03:56.01 that encode the three genes
00:03:59.17 necessary for metabolizing galactose.
00:04:01.11 And the idea was to take the two DNAs
00:04:03.26 and join them together.
00:04:05.22 And we had to develop a way
00:04:08.04 to join DNA molecules together,
00:04:10.11 and we employed what was already known as
00:04:12.29 cohesive ends in the bacteriophage lambda.
00:04:15.27 The bacteriophage has these ends
00:04:19.03 which are cohesive,
00:04:20.24 that is, the two ends can be joined
00:04:22.25 to each other to form a circle,
00:04:24.13 or they can be joined to different molecules
00:04:26.03 that have the same kind of ends.
00:04:27.19 And so the idea...
00:04:29.11 could we synthesize synthetic ends
00:04:31.12 onto the two molecules we wanted to join?
00:04:33.19 And that was already known how to do it.
00:04:37.00 There was an enzyme that would polymerize A or T
00:04:40.09 onto the ends of one molecule,
00:04:42.13 and A or T on the other,
00:04:44.29 so you have two molecules,
00:04:46.23 one which A ends and T ends,
00:04:48.24 and if you mix them they come together.
00:04:51.08 And that was the scheme we set out to do,
00:04:53.05 and that turned out to be pretty straightforward,
00:04:56.09 I think it was actually done within less than a year,
00:04:59.05 and so we were able to make the first recombinant DNA,
00:05:02.18 although it wasn't called that at the time.
00:05:04.25 The first recombinant DNA molecule
00:05:07.05 was part SV40 and part lambdadvgal.
00:05:11.03 And the idea was to be able to
00:05:13.19 propagate these molecules in E. coli,
00:05:15.20 and maybe make mutations in the SV40 sequence,
00:05:19.04 but also to transfect them into mammalian cells
00:05:22.21 and to test whether we could get expression
00:05:24.20 of the exogenous genes.
00:05:27.08 So, that was the first idea
00:05:30.15 and the first fulfillment of that idea,
00:05:33.00 and that of course led, ultimately,
00:05:35.05 to the evolution of the whole recombinant DNA technology,
00:05:38.11 except that it became much easier
00:05:40.21 to join DNA molecules together
00:05:43.01 through cohesive ends created by restriction enzymes.

This Talk

Speaker: Paul Berg

Audience:

General Public
Educators of H. School / Intro Undergrad
Student
Educators of Adv. Undergrad / Grad
Researcher
Educators

Recorded: March 2011

Talk Overview

Dr. Berg reminisces about the ideas and experiments that led to the creation of the first recombinant DNA molecule.

Speaker Bio

Paul Berg

Paul Berg joined the nascent Biochemisty department at Stanford University in 1959 and has remained there since. Berg’s early work led to the discovery of the aminoacyl tRNA synthetases and tRNAs and their role in protein synthesis. Berg was a pioneer in developing recombinant DNA techniques, work for which he shared the 1980 Nobel Prize… Continue Reading