Oliver Smithies:[00:00:00] This is some correction to page 75 of Book m, where I’m talking about DNA prepared on two previous pages and then looking at the color of the plaques. The five blue plaques were obtained. That means that the plaques contained the lac gene, which was an indicator, then, that could be used to identify a plaque. Because once that gene was transcribed, then a plate with the suitable indicator would give a blue-colored plaque. And a white plaque would not have the lac [00:01:00] gene in it. And this whole idea was still to overlap to use sticky ends to hybridize to a cDNA of hemoglobin next to the lac gene and try to use those two things to isolate the hemoglobin gene or a clone of cDNA, presumably, of hemoglobin. But anyway, on page 75 it talks about five blue plaques, which means that they contain the lac gene. All should be [00:02:00] globin-positive, if the procedure that I was hoping to use would work.
And then it refers to a later page, July 1st. Look at that page again. And so July 1st is talking about the results from [m‑75?]. “The five blue plaques were purified and regrown. Four grew. Two were white and two were blue.” On plaque hybridization, the two whites were globin-positive. And [00:03:00] so I was getting some indication of getting globin cDNA-containing bacteriophages.
So this is Book n, starting September 25th, 1976, and going on to sometime in November of the same year. So it’s a fairly short period of time but a fairly hefty book. It begins with characterization of nick-translated [Hb3?] DNA. This is a phage which contains some cDNA from some sequences that are complementary [00:04:00] DNA, made from messenger RNA, I presume, as later on becomes obvious. Just in clarification, when we finally succeeded in cloning human globin genes, we used as a probe complementary DNA made from mRNA, the human mRNA corresponding to hemoglobin and mouse mRNA corresponding to hemoglobin. So these were gifts of mRNA from various people, that are referred to in the papers that were later published. But anyway, going on with this, characterization of Hb3 [00:05:00] Charon DNA — fourth round, I think it is. This was labeled with 32P phosphate. (pause) We used to make the labels from carrier-free 32P and end-labeled — and used this to make [00:06:00] dGTP, dCTP, etc. — with phosphate — 32P.
We had some rather exciting times. There was one time when we discovered a large signal from the Geiger counter in the corridor of the floor where Fred Blattner and I worked. This was one weekend. And so trying to find what had happened. We used the counter. We were a little nervous. We used the counter and we could see that somewhere on the floor was the material. So Fred used a rather thick sheet of plywood, behind which we could hide, as it were, from the 32P. [00:07:00] And we walked down the corridor with the Geiger counter occasionally in front of the plywood, until we reached the area where the counts were very high, and then lowered the plywood sheet onto the ground, so that it now covered wherever the contaminant was that was in the corridor. So we’re now free from radiation ourselves and the very high-radioactive material is behind the — or underneath the plywood sheet. And so we started to move the plywood sheet down, to get to the point where it would expose the contaminant. And a very strange thing happened, that, as we moved the plywood, we moved it [00:08:00] past the place where the contaminant could have been and there was no contaminant there. But contamination was still on the plywood. And we eventually found out it was a piece of cotton wool, which stuck to the plywood. And the cotton wool had been contaminated with the carrier-free phosphate during our making of the radioactive nucleotides. (laughs) I remember that we were somewhat nervous — [or been?]…
But anyway, we are using this sort of material to label wh– So it will be dGTP or dCTP or whatever we needed. So that was a nick translation of a hemoglobin-containing phage, on page one.[00:09:00] So, for example, a remake of the probe, on Monday, September 27th, page five, illustrates a little bit about what we were doing. We’re taking dATP, dCTP, dGTP, and dTTP, for 2.5 microcuries of each. And then added the phage with the hemoglobin cDNA in it and nick translation buffer — and made a probe, then, called n‑5 probe, 1.8×106 counts. [00:10:00] So we now have a probe.
Bam digests, on the following page, digesting the DNA with a Bam buffer. So the DNA was digested, m‑93 DNA from the phage, about two milligrams of DNA. And n‑5 radioactive Hb3 was the probe. And both digested. And then n‑7 [00:11:00] Bam is the result. And one can see a labeling of some specific bands and quite a smear behind it all. With the comment that “The provisional scan looks good, or at least much better. The counts go back all the way to the origin and are not more than 50% up front.” But there were bands in the material. So did label some specific bands, as well as a general smear.
The next page contains a reprint of an article from Tom Maniatis, the “Nucleotide Sequence of the Rightward Operator of Phage Lambda.” [00:12:00] So why that is here is not immediately clear, except that it contains, I think, the [tie fran?] to 3’ extra-nuclease nick translation, with a diagram as to how this works. You make a nick in the DNA and then extend it with radioactive material. And that material can then be used as a probe. And we used it a great deal, extra-nuclease nick translation.
So continuing. And DNA preparation again — with RNAs and Pronase. So [00:13:00] the cells — which cells, not immediately clear — were washed with [pebia?] and Pronase was added. And RNAs… And a comment at the bottom, “Looks very promising. Almost no goop. Water clear and very viscous.” And thinking about adding Pronase, then, as well, to get purified DNA, presumably from the embryonic cell culture, as is suggested by n‑13, “Emb. –” for embryonic — “DNA extracted,” etc., “with phenol.” [00:14:00] Ending up at the end with about 274 milligrams per ml of n‑13 DNA, page 17, October 1st. (long pause) [00:15:00] (pause)
Trying to do gel hybridization, with a method that Fred had developed, that we’ve talked about. (pause) [00:16:00] And the gel was from the beginning of the book, the so-called n‑1 gel, which had human DNA ‑‑gested with R1. And that gel was being used in this experiment. A comment… No, forget tha‑‑
More gel tests, on Thursday, October 14th, where now I can see better what’s going on. Because there’s a good image of the big smear that one gets with a human [00:17:00] DNA digest, and along with the bacteriophage. And an attempt at autoradiography. But it’s a pretty messy gel. Another gel was run — and photographed — with ethidium bromide and then dried down — [and now it’s lying?] — and exposed to the Charon [lac‑?] DNA. So it’s a somewhat obscure result but clearly not very satisfactory.
A new gel idea, on page 31, being talked about. [00:18:00] And a pickup on Ig cloning, on page 33. (long pause) Left and right arms of lambda phage, being talked about a little — Alan Deutch. This is an experiment related to Alan Deutch, on page 33 — [00:19:00] a digression. (pause) And continuing in the same way, and several pages. (pause)
Trying to add a string of dTs or ‑As, with the terminal transferase, on page 41. It becomes a little [00:20:00] clearer what’s going on, that either dATP or dTTP was used with terminal transfera‑‑ to generate sticky ends which had a string of As or a string of Ts. With the comment that “The dTs are easier to add under our conditions. The dAs are poor, not sufficient. And therefore, repeat the dA experiment, with –” it looks like — “with cobalt.” But a reference to nucleic acid research.
Tests of the latest gel — the latest digest, on page 45, [00:21:00] October 25th, continuing with dA and dT with cobalt. Terminal transferase, on page 46. (pause) dATP revised to cobalt, (laughs) on page 49 — dATP. Cobalt-containing buffer described.
Looking to have added [00:22:00] these tails, on page 51, October 26. “90 minutes [dTM?]. 270 minutes.” And then, “dA, 95 minutes” and “dA for 270 minutes.” Looking to incorporate tails. A conclusion — “The dT reaction mixture contains a bad nuclease contaminant.” (pause) So not completely satisfactory.
And summary of the terminal transferase r‑‑ [00:23:00] on page 53, with a comment on one group of them, “Abandon these material.” And the others were — couple were checked. With a big exclamation mark, “These should be multiplied by [1 over the relative concentration of the end?]!” etc. So the conclusion being to repeat the experiment.
And then that is then taken up, on page 57, repeat of the [dTn?] labeling in cobalt. And in this case, [00:24:00] no degradation. “Addition of dTTs is detectable but not great.” So something has been added to the ends. All a matter of getting the right label for probes. (pause)
Repeats, and R1 plus or minus ligase, on October 29th. “Conclusion: (laughs) Insufficient rigorous conditions for the dialyzed sample. Repeat,” [00:25:00] question, “Due to magnesium? Or try EDTA,” etc. Continuous repeating of the experiments — and looking at the digests. Nicking tests, with sodium hydroxide, on November 2nd, page 77. Zero, 5, 10, 20 minutes, then sodium hydroxide. “Conclusion: No nicking and no ligation.” [00:26:00] (pause) Trying to hybridize tails, etc., and no evidence of ligation, on page 79.
Continuing with the problems. Tests of 37° ligation, various ligases, and three EcoR1 preparations, on Wednesday, November 3rd, page 83. And a gel, on the following page. [00:27:00] Talking about the ligase and degrading of the DNA. So I have as a R1 alone, without any ligase — and quite nice bands. But as the ligase is added, the bands disappear. So the conclusion is that “Lot 10 ligase definitely degrades at longer time.” Of course, these days, very few purified materials could be bought. Most had to be made.
And, for example, on Saturday, November 6, the aim was to “Find the minimal ligase and R1 concentrations which would give complete reaction in 1 hour at 37°.” [00:28:00] And examples of the result, on page 89, with the conclusion that “[R1 diluted over 45?] is still an excess. [Ligase diluted over 18?] appears to be below the optimum.” It appears that single-stranded ligation is as far as it can go, and only about 20%.
Trying to get a phosphatase-free R1, etc. Difficulties of obtaining the good enzyme. [00:29:00] Quite difficult work. (pause) Page 95, it looks like HindIII plus R1 and HindIII plus ligase and HindIII plus R1 plus ligase, etc. With the conclusion that [00:30:00] “[R1/200?] is still plenty of R1. And the ligase is still independent of R1 level. Test terminal transferase.” Continuing in the same vein.
Page 101 has a stained gel conclusion. This is R1 and ligase, terminal transferase and HindIII buffer, in sodium hydroxide for different lengths of time, it looks like. Twenty, 60, 180 minutes in sodium hydroxide and 20, 60, and 180 [00:31:00] minutes of native DNA. And the same gel conclusion is that “The [t/20?] and t/180 are all the same after NaOH.” And they’re in bad shape. And the autoradiograph completely confirmed — “The heavy label on the 27% piece when native and none when single-stranded.”
Difficulties in making radioactive probe. Continuing in the same vein, [00:32:00] 105, 107. Various ideas to try to improve the labeling. For example, time test on Bob Wells’s terminal transferase, at 1X, Tuesday, November 16th. One depended upon making or borrowing or scrounging from different people, reagents.
Well, Wednesday, November 17th, a rat experiment. “Check of [DAT+?] versus DAT‑.” [00:33:00] Can’t believe that’s really r– It looks like rat. But what has rat to do with it? Haven’t the faintest idea. So Harvey Faber’s material. (pause) And it’s talking about the gut passage. So that must have been the DP50 supF experiment. That “Harvey’s [Hf 9, 91, and #4, all one, t=3?], was gut passage of DP50. 8 non‑red DP50 colonies could be [00:34:00] found. Harvey counted 9 but one looked like junk,” etc., “All 8 colonies were DAT‑, [GAU‑?]. The controls were DAT+, GAU+. And DP50, the real stuff, was DAT‑, GAU‑. So the results are completely unambiguous,” that the material that was being measured on the gut passage was the correct mater– And that can’t be rat. It must be… (laughs) I don’t know. Anyway, something about rat. Oh, yes, it evidently was an experiment with rat. This was passing through a rat — uh‑huh — not through a human one. So, “The [00:35:00] colonies on Harvey’s plates are large and 3 were sectored,” etc., etc., “This represents a test on the most positive animal.” There were only two. So this was a test in rats or fecal transport — perhaps before the human one yet. Yes, it’s part of the whole test. So that is understandable. It really was a test in rats.
And the next test is what the effect of — in sewage and tap water. So these were repeats of experiments incubating the DP50 supF in sewage and tap water. (pause)[00:36:00] What were called [AB?] plates. Had galactose in them, eosin, and methylene blue and diaminopimelic acid, thymidine, biotin, nalidixic — from Millipore, etc. Bacterial plates. And the plate that later became the one that I remember using a great deal was NZY, which had [00:37:00] N‑Z‑digested material, that one could buy, and yeast extract and sodium chloride. And ZY plates — used to be called the old L plates.
So here we were testing DP50 in sewage and tap water — repeats of these experiments. Protocol being written on the following page, to “Take raw sewage and Millipore-draw sewage and fresh tap water and add overnight cultures of [DP50.F?].” This is the first time [00:38:00] I remember seeing the F there. Made various dilutions, etc. And, “At 24 hours, dilute.” [Nuclear?] tests. And they were called SS, for sterile sewage, [TAP?], for tap water, and [RAW?], for raw sewage. Set up the tests on Friday, November 19, with a result on the following page.
Some tests would have been done with 1100.5, [00:39:00] as well as with DP50.F. Provisional look, on Saturday. “1100‑5 can’t be continued, due to mixed colony. Scrap it and continue with DP50 supF.” Twenty-four-hour titers. “Tap water down to about 1/10th in 24 hours. Sterile sewage down to 1/20th in 24 hours. And raw sewage down to about zero in 24 hours.” So raw sewage was unfavorable.
Continuing in that way. A 72‑hour test, on page 129. “Conclusion: DP50 supF decreases to about 1% in 72 hours in raw sewage and decreases to about 0.01% in 72‑hour sterile sewage.” So it’s presumably growing in the raw sewage but not growing in the sterile sewage. Some metabolite may be being produced in the raw sewage that allows DP50 to grow. And, “Down to about 0.1% in 72‑hour tap water.” So it was making reasonable sen‑‑
These experiments were repeated, on Monday, 22nd, repeat of the sewage and tap water tests. [00:41:00] With a note on what — “1110.5 [F‑, Endo‑1‑ prototroph, Anal-resistant?].” Twenty-four-hour tests, on November 23rd. And a comment that “Sterile sewage grows gal+, DAT+, an increase. SS holds [00:42:00] gal‑- DAT‑ steady.” So samples got from material in the sewages make a difference.
Here’s a repeat of feeding tests now, in this case, of Fred’s experiment. And the fed material. “9.4×108 of gal+” is on the left-hand page, 134. And that’s the typical number that we saw — and the page from that cover of Science. And we’ll just pause a moment to check that. (break in audio) Just in clarification, a little bit, of these experiment, the final publication talks about [00:43:00] “9.4×108 of 1100.5 was fed in 250ml of milk to three humans.” And here is the titration, on page 134, of 9.4×108 gal+. So this is experiment was done on 1100.5. That was a normal bacteria. So that was the normal one. So this actually is — Tuesday, 23rd of November, page 135, in Book n — it does describe part of this experiment. And it’s fun to see [00:44:00] the 9.4×108 mentioned in both places, that the… But the mixture also contained 3×1011 of DP50 supF. I’ll read that a little more, just to be sure. (break in audio) The mixture was a mixture of 9.4×108 of the 1100.5, which is a more robust bacteria, compared with 3×1011 — that’s at least 100 times more — of DP50 supF, fed at the same time. So I looked for the… As it says here, the ratio was 1:300, [00:45:00] on page 134. Because the titer is there, given, of the — presumably the titer of DP50 supF. Or I should be able to see it. (long pause) No. It is there on this page, in [00:46:00] fact, on the left-hand side. It’s 9.4×108 of gal+ and 3×1011 of gal‑. And that’s DP50 supF. So DP50 supF was 3×1011 and the 1100.5 was 9.4, which is a ratio of 1:300. So this is the page containing all of the input material for Figure 4 in the paper that was published with a cover of Science, and April 8th, 1977. It was about four months later that the paper was publi– But there’s the input material, on Tuesday. Was fed to three people, Bill Williams, myself, and Fred Blattner. And then [00:47:00] here was the material obtained at 24 hours and 48 hours and 720 hours, being — sorry — and 72 hours, being quoted.
A summary of a t– That was the next page — doesn’t have the summary of those results. It’s a summary of the raw sewage and the tap water. We haven’t got yet to the page summarizing the results on the humans. Summary of Sewage 2, on page 139. Summary of Sewage 1, on page [00:48:00] 141. Phage test on sterile sewage, on page 143. Protocols for preparing lambda phage by phenol extraction, Fred Blattner, on page 145.
And it looks as if I don’t have the results of the human passage in my book. I have the input but not the result. But I’ll look back on this again and try… So let’s go back, Jenny. (break in audio) [Is?] reason for a little bit of uncertainty. The [00:49:00] results on page 135, the initial material, are not my titrations. They’re Fred’s experiment. And so it’s just summarizing what the material was. It’s not actually the titration. So on page 134, the numbers are correct but they are from other persons’ measurements. So although I knew very well what was fed, etc., and the ratio 1:300, I didn’t actually make the mixtures or titrate them.
So where were we at, Jenny? So continuing the book, on page 144 and 145 I have a preparation of DNA from lambda phage [00:50:00] biphenol extraction protocol from Fred Blattn‑‑
And on page 147, the following page, I have a new genomic cloning scheme. This depends upon having the left‑ and the right-hand arms of the bacteriophage with RHSE — in other words, the right-hand with a sticky end — minus and a left-hand sticky end with a plus. “Any cut but not HindIII or R1. And then make +/- beads. Make beads in which the two arms are joined [00:51:00] to a bead,” and, etc., joining the left and the right arms to get a complete genome again. And try to insert, presumably, cDNA in between the two. Yes. But step one is prepare right-hand sticky end and left-hand sticky end “and make beads with the ends on them. And then make, from Charon phage 3A, minus lac, left-hand arm and right-hand arm. And check that those two steps [will?] clone when one arm has a HindIII [00:52:00] end and the other arm has a HindIII end. And –” no — “the other arm has an R1 end,” and then that, if you add some material that has H and R ends, that it will clone, to pick up… So this is a way of capturing a piece that is H on one end — HindIII sticky end on one end and R1 on the other. And with a comment that “This has no competition except a legitimate recombination.”
And the scheme continues on the next page, 149. “The main difficulty is proceeding further without a probe.” So the scheme [00:53:00] of Fred and Harvey is better. And gives a little outline of Fred and Harvey’s scheme.
More thoughts, continuing the same line of argument, on page 151, as what is needed to get what we’re trying to get, with C‑ and G‑tailed material, terminal transferase C, terminal transferase G on the incoming DNA and terminal transferase Cs on the phage, so that you get overlap, with [C, G?], and both ends. And cloning to the right phage and using a cDNA probe.[00:54:00] Talking about the enzymes Hae and [Psd?], H‑A‑E and P‑S‑D, to get overlaps, with [usual?] alternating enzymes, etc, etc. And that’s the end of this book, Book n. [00:54:28]