Oliver Smithies:[00:00:00] So this is the start of Book m, March 1976, going through to September of the same year. And again it [was?] here, transitioned to more understandable methods than the sticky-end method. So begins with EcoR1 tests with Charon 3, one of Fred Blattner’s safe viruses — bacteriophages. Page one, tests of this and the results of a digest — and the fragments obtained. [00:01:00] Clearly, there were four of this fractions. And the map is shown, and what is expected.
And the EcoR1 with HindIII digests, on the following page. And these are becoming much more familiar patterns, as far as I’m concerned — to the ones that became deeply ingrained in one’s mind. EcoR1 plus or minus HindIII, plus or minus SE, whatever SE is. [00:02:00] (long pause)
We have to ta‑‑
(break in audio)
We start again. [00:03:00] This is the beginning Book m, with EcoR1 plus or minus HindIII digests, plus or minus sticky ends on chromosome 3. SE. Digests. And then going on to a protocol obtained from some kind person on how to use terminal transferase to label the ends of DNA, on page six. Still sticky ends being considerably thought of. [00:04:00] But beginning to make some sense, in terms of maps and DNA gels.
Question of ligations and what concentration of buffer is needed — or what concentration of fragments is needed to get the best ligation versus circularization of fragments. There was an equation which we had some enjoyment with called the Stockmayer equation, to calculate what is the optimum concentration of DNA so that end-to-end ligatio‑‑ will [00:05:00] occur rather than circularization, etc. Fred Blattner had — he named a little program for the Stockmayer equation after his son, whose name I don’t remember but it may come up, as we read through the book. But we had some enjoyment with a little computer program to calculate Stockmayer concentrations. So here, on page 11, talking about an improved construction.
Ligase tests, April 14th, with Charon phage [00:06:00] 3A, which later was used a great deal, this particular Charon phage. So talk about it. And an R1 digest of it, on page… No, that’s not quite true. m‑15, control DNA was digested — on page 15. Rerun on the following page, a quite reasonable gel. Repeating the digest and the gels — respectable, on April 19.
Uh! Now then [00:07:00] the terminal transferase talked about again, page 21. “Use about 0.1 mg/ml and,” etc. And the Rabbitts method of using terminal transferase. So presumably, that was the protocol that had been received from Rabbitts. There still wasn’t any handbook on how to do experiments with DNA yet available. (laughs) April 20th, sticky ends again. And not very satisfactory.[00:08:00] And the following page, careful scrutiny gives a map of Charon 3. And suggesting that “A long gel will resolve HindIII, R1,” etc., etc. And a gel testing m‑13 control DNA, m‑15 R1 digest, R1 and HindIII digests. Clear evidence of ligation. “Estimate about 80% ligation,” of this material.
Terminal transferase, [00:09:00] page 27. Reverse transcriptase. First mention I’ve seen of that — on Charon 3A. And these pages are actually flagged by label on the top of the — attached to the page. And so the first one was “Terminal transferase,” on page 5 — “Ligase of Charon 3A,” on page 13; “Terminal transfera‑‑ of Charon 3A,” on page 27; “Reverse transcriptase of Charon 3A,” 29; “Charon 3A lac hemoglobin transfection,” page 41; [00:10:00] “Charon 3A lac ligase and terminal transferase,” on page 45; and “Charon 3A reverse transcriptase,” on page 49; with “Charon 3A terminal transfera‑‑ #2,” page 51. So these pages are flagged as being important landmarks.
So we’ll go back to progressive state. (break in audio) So now continuing again with the regular progression of pages. Page 27 is a terminal transferase reaction. “2,000 units of 84,400 units/ml.” [00:11:00] A label from… “See Harvey’s notes,” it says, “of the enzyme.” But I don’t have his notes. “Clear terminal transferase from…” I don’t know. Reverse transcriptase method considered and used, on page 29.
The experiments are beginning, to try to label the DNA in these different ways. The result, on page 33. [00:12:00] Then “15 R1 labeled” and with a circle, saying, “m‑31 cDNA,” complementary DNA. So this is beginning to make labeled probes with reverse transcriptase. A fairly decent autoradiograph and ethidium bromide-stained gel. Terminal transferase number two experiment considered, on page 35. [00:13:00] Trying to get good labeled probes. (pause) Recheck on m‑31 cDNA, complementary DNA, product, on page 39. A conclusion — “The proper sample of m‑13 cDNA is well represented in the ligased part. Can estimate from two ends on lac piece,” etc., etc. [00:14:00] So a protocol, on page 40, from Tom Broker, on renaturation kinetics.
Beginning to understand melting of DNA. So here, on Thursday, April 29, “11 plaques were recovered, Harvey Faber. Subsequent hybridization with 32P cDNA shows 5 [00:15:00] out of 11 are tentatively globin-positive.” This is starting with m‑39 mix. What is m‑39 mix? Page m‑39, it was cDNA — for… Not at all clear where the hemoglobin phages — or ph‑‑ is coming from. But my memory is that they came from Tom [Maniatis?]. But I haven’t [00:16:00] found any reference to that.
So let’s see if it makes sense on Friday, April 30th, page 43. “Summary of procedures and comments for the next run. — Ligation, Charon 3A, KH803 DNA, R1 buffer ligase, and R1 digestion.” Terminal transferase reaction. And terminal transferase and reverse transcriptase. But at this point, nothing containing any reference to hemoglobin.
Labeling again, on page 45, (phone rings) Saturday, May 1st. [00:17:00] (pause)
Jenn, you have a call on 16, Jenn, 16.
So on Tuesday, May 4, page 47b, for some reason or other, (laughs) instead of 48, “Gel test on terminal transferase products. Results are very clear. m‑45 dATP terminal transferase is fine.” So we can label. [00:18:00] And it’s cDNA. So this is learning to label bacteriophages in different ways. Harvey Faber is mentioned here, on page 49. “Harvey before received dNTP from radiopharmacy.” Poly(dT) tails being added to Charon 3A cDNA, on page 53. [00:19:00] Tests of annealing. (pause)
And DNA checks by the electron microscope being considered, on page 59. And continuing to think about labeling, with dCTP and dTTP. With results of electron microscopy sketched, on the pages around about Monday, May 17, electron micrographs with sketches of EM results — no images. But quite a lot of pieces, and 11.16 and 30.73, 29.5, 30.9, etc., etc. So seemed to be able to look at the fragments reasonably well in the electron micrograph. “Provisional conclusions,” on page 69, that “The ligation was,” query, “poor? More likely nucleases eventually broke up and nicked (background conversation) the material.” But this is a result of looking at the [00:21:00] DNA in the electron micro– “No ends and branches,” commented, on page 68. (pause)
Labeling, here. Here is a mention of hemoglobin, [00:22:00] again rather obscure, page 75. “m‑73 DNA and m‑63 DNA,” etc., etc. But underlined, at the bottom, “5 blue plaques. Makes sense. All should be globin-positive, with two genes per [P?], due to trapping of large DNA in the precipitate.” Again, not very clear what was going on.[00:23:00] (pause) Thursday, June 17th, with (laughs) a little note, “Happy birthday, Nancy Exley.” My sister’s birthday. Preparative electrophoresis for a lac piece. And a diagram of the gel, in a column — in a tube, that is to say. Trying to purify [00:24:00] the material.
Larger-scale preparative electrophoresis, Saturday, June 19th. (pause) And June 21st, lac recovery attempt. More Charon 3A for lac, on June 22nd, with a rather more interesting set of gels, on page 90 versus 91, looking at Charon hemoglobin [9A?] as [00:25:00] a clone, hemoglobin 3A as a clone, hemogl‑‑ [Hb?]… This is [Hb‑‑9A?], Hb3A. “Contains duplication.” (long pause) Beginning to wonder if this “Hb” really is hemoglobin. Because it’s talking about comments on m‑thir‑‑ on page 91. “Best bet is Charon Hb3A. [00:26:00] Next is Charon Hb9A.” (pause) Quite a nice gel, on page 90. But what it all means is a little less clear.
(laughs) Page 93, Wednesday, June 23rd, my birthday, and [of?] my twin. So, “Happy birthday, RWS,” Roger William Smithies, my brother, mm-hmm, which would have been… What [00:27:00] year are we in? What year are we in, Jenny?
Doesn’t say. Oh, here it is, ’76.
So, ’76. Been my 31st birthday. Seventy-six, ’76. No, not my 31st. Seventy-six would have been my 51st — 51st birthday. So here I am at 51, on June 23rd. Lots of digests. (pause) [00:28:00] (pause)
So continuing with this saga. New idea for extending the clones, on June 28th, page 95. And page 97, “After talking to Dave McKean and Fred Blattner, better to pre‑purify [00:29:00] [CCC?],” etc. (pause) These are… (pause) [00:30:00] (long pause) So [00:31:00] continuing with the result from m‑75, although I don’t understand them, that “Five of blue plaques were purified and regrown,” etc., “They should be single-clone pieces with R1 sites. Two white were globin-positive.” These are [quaternal Egwotz?].
Page 101 I do understand. This is two strains obtained from Roy Curtiss, to… Roy Curtiss was developing his bacteria that were very debilitated as a result of mutations, that he called the [00:32:00] Curtiss or Chi bacterial strains. Because people were still in the phase of not being sure that it was safe to clone human genes, except under very restricted conditions, which is what Fred Blattner was dedicated towards doing. And so he telephoned Roy Curtiss, asking for a bacterial strain that was weaker than normal, but to increase the safety level achievable with his bacteriophages. So Charon phages were going to be grown on a defective E. coli strain, to increase the safety factor. And he tried to get hold of Roy Curtiss and didn’t succeed, and instead [00:33:00] talked to Dennis Pereira, who was Roy Curtiss’s postdoc at the time or graduate student. I don’t know which. But then Dennis Pereira sent his strain, which was called DP50 supF, as I recollect. Anyway, it was a defective bacterial strain, that required diaminopimelic acid to grow and carried a suppressor gene, Suppressor F, which would allow Fred’s bacteriophages to grow. So Fred’s bacteriophages had various chain-termination mutants — and various places — and needed a [00:34:00] suppressor tRNA in order to grow. And DP50 supF — or DP40 supF — we’ll see the name in a little while — was obtained from Dennis Pereira. Was some controversy about that, which I think I’ve mentioned earlier. But if not, we’ll talk about it at a later stage. Because Roy later on thought that this was inappropriate, to have gotten it from Dennis Fereria without talking to him, which is probably correct. But anyway, two [dap‑, samRd‑?] strains from Roy Curtiss, to test for Charon growth and for transfection, on page 101. [00:35:00] It is DP50. Because, “All Charons grow well on [B50?],” which became later called DP50, because it was Dennis Pereira, strain 50 — and had supF in it.
(laughs) This is at the beginning of the test that was finally done of the… Because it was needed to show that passage through the human intestine would result in very serious decrease in these bacteria now. So bacteria, as I remember the cover of Science there — has a picture of Fred’s Charon phages and the bottom of the cover has a comment that 109 bacteria… And I’ll get the exact quote. It’s in the eventual publication of the “Charon Phages: Safer Derivatives of Bacteriophage Lambda for DNA Cloning,” which was published on April 8th of 1977 and made the cover of Science. Fred Blattner is the first author and Bill Williams is second author. I’m at the end. [00:37:00] But the critical sentence is that… To test the fecal — the passage through the intestine. “9.4×108 of,” the strain, “1100.5 was fed in 250ml of milk to three humans.” And the three humans were, in fact, Fred Blattner, Bill Williams, and myself. And we all survived the test. But here, on page 103, is the beginning of setting up that test. Because it’s talk about all the Charon… “Charon phages grow well on B50,” which, in a couple of pages, [00:38:00] becomes called DP50 supF. And here, comment three is… Well, let’s start at the top. “All” Charons “grow well on B50,” comment one. Comment two, “Test at transfection,” and “Result was OK.” And, “A rough test of the fecal pellet handling.” How to homogenize fecal pellets was talked about. Plates were made, etc., etc.
Ed Sheldon — presumably Ed Sheldon… Let’s just see. Yes, it was indeed Ed Sheldon — set up fecal pellets, [00:39:00] plus, etc. — plus B‑f‑‑ with B50 “in future to be called DP50.” That name was approved by Roy Curtiss.
And some testing of sensitivity — UV — of DP50, on 107, etc. — continuing to investigate some of its properties. On page 111, chloroform on DP50. Difficulty in lysing with chloroform, because of the sensitivity of the bacteriophage. [00:40:00] And with a result that “Unequivocally, chloroform for 10 minutes with two shakes does reduce the titer appreciably.” So DP50 could stand chlorofo– Further killing tests of Charon 3A and DP50, on page 115. And a little bit more precise statement, on 114, “Chloroform decreases the titer to about 80%.” Chloroform and octyl alcohol, I presume, “increases the titer to about 500%,” and “Trichloroethanol [00:41:00] decreases it to 10%,” etc., and diethyl pyrocarbonate, “DEPC decreases it to about 10-3%.” So killing tests on DP50.
DNA transfection protocol, September 2nd, 1973, written down. KH502 standard was 2.9×10-6. DP50 with lysozyme, that’s the same as the standard, 2.9×10-7, and without lysozyme, 1×10-6, etc. And with a note that “When [00:42:00] lysozyme is added to DP50, there’s trouble with re‑suspension.” So lysozyme and DP50 supF are a little bit incompatible.
“We can detect chromosome 3 lysogen,” on page 121, “by release of the plaque color and the color of the column.” I’m forgetting there is a lacZ in these phages. And that’s where the color of the column is coming from. I’d forgotten that there was lacZ and we could therefore use plates with the indicator for activity of lac [00:43:00] and get color indicating that the material there could express that gene. So that would be explaining what shouldn’t have been a mystery, the color of the phages, that had puzzled me — color of the column. This is an indication of whether they were expressing the lacZ gene.
Test, on page 123. “Conclusion: DP50 is not sensitive to 0.05% deoxycholate.” [00:44:00] New enrichment schemes, on page 125. A digression of going back to separation of chromatin containing bromodeoxyuridine in one or both strands of cDNA, which was Lorne Taichman and Liz Freedlander’s paper, in 1976. And then I’m helping Liz, evidently, with making nuclei, with notes on what the protocol should be.[00:45:00] And page 128 and page 129, talking about repairing EcoR1 sites from a cutout of a fragment made with EcoR1 and then to lygate it to an EcoR1 fragment with some other way of getting rid of the need to use terminal transferase. [00:46:00] And continuing with that general idea, in the following pages.
Back to DNA and nuclei, from S05 — I think S‑‑ no, 563 embryonic cell, on page 135. (pause) Two recipes for making DNA from these cells, Susie’s recipe, page 138, [00:47:00] and Lorne’s recipe. “Susie’s” is Levan and “Lorne’s” is Taichman. And 139, DNA. (pause)
So learning to use diethyl pyrocarbonate and clean things up with phenol and hydroxyquinoline, on page 139. Provisional conclusions, on the following page. [00:48:00] And the use of [Pronea?]. (pause)
Talking now about digest of human DNA, on page 143, September 22nd. “Human DNA can be loaded after R1,” etc., “at 50 mg per slot.” And some examples of R1‑digested material and a long smear of DNA, [00:49:00] typical from a human DNA — and 139 DNA. And 139 is the page of making it. And it is material isolated from cultured embryonic stem cells. (pause) [00:50:00] (pause) That page, 143, the gel shows that it was necessary, after digestion, to have rather a large amount of DNA in order to see the digested product. So the 4X Helling’s gel, 0.75% agarose gel shows the control — is just showing what the total DNA looks like, which migrates only minimally into the gel. And after digestion, the only one that you can see reasonably easily is the one at high concentration. [00:51:00] The others, after digestion, are too spread out to see. But beginning of a good electrophoresis of mammalian DNA digested with EcoR1.
So on page 145, it goes on with this idea of — “Decided to — R1, the whole of the remainder but under more concentrated conditions,” so that we could do in situ hybridization, using either complementary DNA or a clone probe. So the beginning of being able to hybridize EcoR1 digests.[00:52:00] Page 147 — m‑147 — preparation of R1‑digested DNA. And, “Gave this to Ed to try for histone genes.” Because he could try for histone genes, whereas we were wanting to find hemoglobin-containing genes.
Page 148, the Blattner hybridization technique. Was in situ gel hybridization. He almost invented what became the Southern blot method but doing it in gels, without transfer. [00:53:00] But the problem was that, Fred’s method, the material diffused rather slowly. However, he did manage to make it work. But whether it was published or not, I’m not sure. But Ed Sheldon’s method was a better method of finding colonies in a digest. But here is his technique described, on page 148. “For in situ gel hybridization. Ran an agarose gel. Stain it in ethidium bromide. Photograph it,” and then “In [plaquo?] hybridization,” etc. And “in [00:54:00] 1m potassium hydroxide for 30 to 60 minutes, to melt the DNA.” And so on and so forth. And, “The hybridization labeled RNA or DNA over several hundred nucleotides. Must be partially broken up to keep the background down, most easily accomplished by sonication.” But it was a good attempt. In fact, I think I’m an author on that paper describing that, which also includes the Helling 4‑times buffer. And we’ll look that up in a moment.
There’s a recipe for nick translation, which [00:55:00] later became a common method of labeling DNA, to make a probe. “DNA Preliminaries 1,” etc., etc., using alpha-32‑phosphates-labeled dNTPs, of each or of one, and cold others, dNTPs. So one could label one or — of the bases. And in situ hybridization is talked about on m page 151. And nick translation is there. And then [00:56:00], “m‑150,” parenthesis, “[(32P) Hp3?].” Must have been material labeled from a cDNA clone. I think we had cDNA clone from rabbit hemoglobin, if I remember. But we’ll have to check that later. So that’s the end of this book. [00:56:34]