Oliver Smithies:[00:00:00] So this is Book v, beginning Saturday, March 31st, 1979, and going through to August of the same year, now trying to get different clones of hemoglobin DNA. I think this is probably already after the time that Fred and I had decided which way to go. He chose to go on with the immunoglobulin work. Because we’d got an immunoglobulin clone in our first experiment. And I chose to go on with hemoglobin, then, as the alternative. I remember feeling that the problem I had been most interested in in immunoglobulins, which was the antibody variability, had been solved, by [00:01:00] the experiments of — [00:01:05]
OS:[00:00:00] Yeah. And I chose to go with the globin gene, whereas Fred decided to do immunoglobulins. And I was less enthusiastic about that work, because the problem I had been most interested in with immunoglobulins, which was the variability, the main part of it had been solved, I felt, by Susumu Tonegawa and his finding of the joining of the variable and constant region of DNA — to make antibody variability understandable.
So anyway, here we are on page one, partial digests of AB, which is Ann Blechl, Clone 165.12, which is a complete G gamma and A gamma region, [00:01:00] a large piece of DNA having both features of the globin gene and with a preliminary order there already written out, on the page before page one, on the left-hand side, preliminary order of the fragments. With a rather beautiful gel. But the DNA in different concentrations — or, rather, with different amounts of EcoRI in the sample, starting from [00:02:00] 1/1000th-diluted up to 1/10th-diluted of our standard recipe. Where one could see the bands being partial and completely digested — that would appear that complete digestion required a tenth dilution.
And here, on page three, April 3rd, is a repeat of the partial digest and what evidently was used for making a slide, in those days, the stain of increasing R1 with hemoglobin 165.12 and the hybridization [00:03:00] to it with globin cDNA, of… Whether the cDNA was beta globin or gamma globin, I can’t tell from the photograph there. But I assume that… So it was April 3rd. And it was 11 micrograms total of the [ABG‑2?] 165.12 DNA, with the ligase and DEPC and then EcoRI. [00:04:00] The probe is not clearly stated there. Anyway, there was a probe used, which was 32P‑labeled cDNA, which is probably described in the following pages. A provisional map showed that there was 140 kilobases of DNA, a beautifully large piece — and that the hybridizing bands were at 6,900 down to as little as 270 [00:05:00] base pairs, one, two three — six EcoRI fragments, which could be reconciled with the map well. And with a comment, for example, that “This should be complete G gamma. And cut it out for re‑cloning into pBR322.” But it’s a beautiful gel. And the hybridization to it, which is shown on this page facing p. 3, is a very nice hybridization. Altogether, a very satisfactory result. [00:06:00] And so we now have G gamma and A gamma genes both on one clone. And…
And started to map more seriously, on page 9 — further restriction map of 165.12. And then we could decrease the containment rules, if we could show that we knew what the region was. And so we set out to do this by mapping [Pscs, BarI?], BamHI, and [beta‑2?] sites, [00:07:00] to get the proof.
And double digests were carried out April 7, page 11. And another beautiful set of hybridizations were obtained, on the following pages, page 12 and page 13. Very enjoyable to look back and see how pretty those gels are and the hybridization. They must have been very rewarding at the time. Because it certainly is, looking back at them at this time. Although, “[Bagel and hist?] require repeating,” as far as the comment is concerned. But the repeats were made. And hybridization expectations given on page 15, [00:08:00] and a provisional map on the same page, drawn by hand — showing the map and the expectations.
So leaving that topic for the moment, we go back to Xenopus laevis [recicide?] DNA, with a summary of what happens during the development of — or, [00:09:00] no, rather the different cells that can be obtained in whole blood, from small lymphocyte up through red blood cell and up through F, which is perhaps junk.
So re‑cloning into pBR322, with the G gamma gene, as mentioned previously. Aim to use the two-piece partial of G gamma, which should be obtainable from a preparative gel. And then the partial digest is carried out on page 19, and with a conclusion that it’s an excellent partial and can get out [00:10:00] the full-length clone, of 6.9 kilobases, from the digest. The marker [makes?] — as a guide. The slowest doublet should be 6.9 and 9.8 kilobases long. So a preparative gel was made. And the edges were cut off and the sample was prepared from the middle. Though it’s not a pretty gel, it was still possible to get [lydying?] out of it, on page 21, April 19th. And looking at the samples on a gel, [00:11:00] as shown on page 23. But the 8.5 and 6.9 fragments were obtained. The yield’s about 50%, overall. The quality was exactly as expected, which is not to say it was perfect. But it was usable.
A new batch of pBR322 used. Test digests of it. Ligations, again, thinking about Stockmayer [00:12:00] concentration, April 25th, Wednesday, page 29. (pause) Happy with the concentration proportions, on page 31. But, “The proportion of fragments was close to ideal and the ligations are very good. Paula obtained the desired [transformance?].” So shows the pre‑ and post-ligation and the clones made from that.[00:13:00] Going back to Xenopus laevis. Xenopus laevis 5.8S and 28S ribosomal RNA — and 8S RNA. And I get 4.95 base pairs for the 5.8S, the 4.8‑KB material. Anyway, slightly different [00:14:00] sizes. So Xenopus laevis ribosomal RNA tests.
And a hybrid plasmid talked about, on Sunday, April 29. I’m wanting to put A gamma 5’‑two-thirds in front of Xenopus laevis 28S RNA. The purpose of this experiment is not immediately clear, to take the 5‑kilobase fragment and the 2.7‑kilobase fragment. Two-point-seven-kilobase fragment being from A gamma [00:15:00] and 5’‑two-thirds of Xenopus 28S RNA. We must — thinking of some sort of transcriptional tests. (pause)
And had too little enzyme, on page 36. And on page 38, overshot the [00:16:00] other way. Try to find the right concentration of enzyme to digest the Xenopus DNA. (long pause) [00:17:00] Rather complicated set of thoughts, in these experiments. But anyway, trying to get the digestions to work. R1 digests, May 3rd, Thursday, again. “R1 digests again, again!” exclamation mark. With a comment that “At 37° it’s evidently hard to get intermediates.” And [K51.11?]… “Evidently K contains a nuclease, since yield is decreasing with an increasing amount of DNA.” On page 41, [00:18:00] time series, on Friday, May 4th. “A big improvement but still not, certainly, the best.” Trying to digest the K51.36 clone.
Shorter R1 times attempted, on page 45, five minutes and five minutes. Preparative gel, from the material made with the shorter times. [00:19:00] “Excellent digest times. Probably could have managed without the gel purification, if not successful.” But anyway, trying to cut out the bands that are needed and staining. And again, a preparative gel, on page 49, May 10th. “Yield better than 50% and purity acceptable,” for the fragments that were cut out.[00:20:00] Getting out the 28S fragment from Xenopus laevis again, on page 51 and 53. Yield is OK, without bothering about precipitation. So preparing the [lens?] that I wanted to clone.
Ligation again, on page 55, with a conclusion that has an incomplete senten‑‑ “Excellent ligation [bonding?]. Transfected with 3ml and got 104 colonies, of which [00:21:00] a,” blank, “cloned positive.” So the answer is not given on that page.
“We need a complete G gamma and A gamma region sub‑clones for sequencing, so run a preparative gel of the whole, to get the fragments of different sizes,” and, of which, they are listed, 1.53, 0.27, 6.90, etc. And then we cloned, from 6.9, the four pieces. Etc. How to [00:22:00] clone the sequences that we needed for sequencing.
Preparative gels, on page 59, May 15th, with the conclusion, “The yields in the separations are close to perfect.” And various purified bands are shown, [B59?] fragments, six‑p‑‑ 6.9, 2.7, 2, 1.60, etc. Each fragment is shown in the gel. And they’re very satisfactory, as it says, close to perfect. Very good purification. And ligating these, on May 17th, with “Stackmayer” [00:23:00] concentrations, instead of “Stockmayer.” We used to think a great deal about that. Set up the ligations. And a high percentage of success in transformation. And looking at them, on page 65, Thursday, May 24th, all the ligations are effective, though somewhat poorer at the small end, as expected. But later results show [the ligand?] is more frequent, with the small end, despite the fact that the ligations didn’t look too good. So before and after ligation, fragments, on page 65, ligating to pBR322.
Birthday greetings, on page 67. [00:24:00] A Gordon conference and vacation, and with Beatrix and Roger. And various farewell. Back to work July 9th.
tk fragment in pBR322, on page 69. Raju Kucherlapati sent 100 micrograms of pBR‑‑22 containing the 3.4‑kilobase tk gene, from Herpes simplex virus. The aim is to put the tk gene and the G gamma gene into a single plasmi‑‑ for transformation tests. So using a thymidine kinase promoter gene from Herpes, in front of the [00:25:00] three-point‑‑ oh, and see — in front of the G gamma gene, to be able to get expression. Beginning to think about how expression is controlled, with the G gamma and A gamma genes.
And so here is EcoRI on the thymidine kinase pBR322 clone, on page 71, July 9th. Conditions are a little too mild. “Try again.” And as before, [00:26:00] “[EcoRI over 40?], 1ml per 10ml of reaction mixture is the best,” on page 72 — with quite nice digestions, 1 over 40 being the one selected. (long pause) [00:27:00] (pause) Trying to get a partial of the tk, rather than complete.
So a bulk reaction, on page 75, with the place “not a very good cut.” But managed to get out some. Repeated it again and got a better [00:28:00] result. A preparative gel. Result on page 77. “The yield and purity completely acceptable.”
Now beginning to ligate the tk fragments to the G gamma gene, on page 79, using Biolabs T4 ligase. And the conclusion, on page [00:29:00] 78, that “The ligation is excellent.” Repeating EcoRI fragments from 165.24, on page 81. The [addition?] was incomplete. “Use more enzyme next time.” Southern blot, on 165.24, for RNA tests. “OK for transfer.” Going to use this B81 R1 digest and test it in different ways — for RNA test. [00:30:00] Gel pieces looked at, on Saturday, July 14th. Partial digests, on page 87, continued.
And DNase on the RNA preparations, on page 89. Decided to try to remove DNA from transcripts that Helena had made from [C165.24?] DNA. (pause) [00:31:00] Using DNase on the RNA preparations, on page 89. (pause) That DNase one looks to have been treated with diisopropyl fluorophosphonate. No, can’t be. DPFF. I have to find out what DPFF is. It’s not important. So I won’t worry about it. But doing DNase on the RNA preparations. [00:32:00] And with a conclusion that “It’s best to use 1/3mg per 10ml of reaction mix for 30 minutes at 37.” So how to digest with DNase to isolate RNA, without DNA attached. (long pause)[00:33:00] So going on with the ligation — one with G gamma and one with A gamma, being ligated into pBR322, to purify the A gamma and the G gamma [edgeles?] separately.
Going back to RNA again, on page 93, RNA test with DNase, etc. “Enough RNA to stain. The DNA works. Check with RNase.”
Going now to transformations again, page 95. [00:34:00] That was the centrifuge. And transformations continued, on page 97, using B91 and B91 G gamma and A gamma ligations. But forgot to add the tk pBR322.
So check on the ligations, on page 99. “Ligations OK. And the RNase test should be repeated.” Continuing in the same vein, for several more pages, including page [00:35:00] 103, transformation of tk G gamma.
And, on page 105, July 20th, got about 150 colonies with 10 microliters of the G gamma transformation versus about 300 on the positive control. And the negative control has nothing. And eventually, at the bottom, Monday, July 23rd, the conclusion that “The results gave 1 positive out of 48. Increase the number [00:36:00] to get more.” And repeated with 192 colonies, gave 10 more, so 11 out of 240, about 5%. And translating the 6.9‑KB piece as a probe, nick-translating. (pause)
This is aimed at — expression experiments, which later on were done with (break in audio) Raju Kucherlapati and Art [Skoultchi?]. [00:37:00] That’s what these preparations were primarily going to be used for. Anyway, they were following up on a request for a collaboration with Raju. “Desirable to make a plasmid –” this is page 107 — “with alpha and beta or alpha and gamma on a single tk plasmid.” And can do this. (pause) That’s a plasmid, looks complicated, combined plasmid with the tk gene, which can work in either direction, going to two alpha [00:38:00] genes on one side and going to G gamma on the other side. (laughs) Using hemin, could then screen for function of hemoglobin — of hemoglobin production. Because there would be alpha chains and gamma chains made by the same plasmid — is the idea, to make both alpha and gamma chains. Rather pie in the sky idea. But anyway, cloning the alpha genes from human DNA was therefore started, on page 109. “Try to get the alphas from Maniatis. Otherwise, can proceed to make them.”[00:39:00] Preparation of BamRI-cloning DNA, on page 113, starting with quite a large amount of Charon 4A [and?] R1 DNA. And then cutting it with Bam afterwards. But the results, on page 112, are that “The R1 is OK. But the Bam needs another check after half an hour more and another 8ml,” of enzyme, presumably.
So a preparative gel, on the next page, the left arm and the right arm of 4A. And a reminder, “In future, use Helling’s X4 [00:40:00] for preparative gel.” Because this one was done with Helling’s-by‑2, by mistake. Helling’s-by‑4 gives more symmetrical bands. The right arm and left arm, on page 117. And then right arm and left arm, from Charon 17 and Charon 4A, being prepared. And gels showing that they are quite good materials.
So combining Bam and R1 human shotgun, on July 30th, page [00:41:00] 119. “Paula tried previous DNA with BamHI plus Jerry Slightom’s EcoRI.” And with various other controls. “The digestion is complete. There will not be a large amount of DNA equal to 13.5 KBP and screen will be easy,” is the conclusion — a little obscure.
So a prep gel made for BamRI, human DNA. Thirteen-point-five kilobase is the [00:42:00] target size. And some markers. And seems like Helling’s-by‑4 buffer, with the places that were going to be cut and images of what was actually made, there. Getting the gel fragments and freezing them, to elute the DNA.
On page 125, Tuesday, July 31st, ligating BamRI 13.5 KBP shotgun, with the right and left arm of phage. “[Cool to any of the arms?]” — and [00:43:00] then packaging — where we’re doing direct packaging of DNA into phages, in those days. And packaging DNA with a packaging mix. So here’s an example, on page 125, as I said, Tuesday, July 31st. Left and right arm. Annealed and precipitated — or just annealed, not precipitated. And then, “10X packaging should give 5mg of phage DNA plus molar equivalents of the target.” Coprecipitate, etc., etc. And a gel run. “The ligation is not [00:44:00] terrific but it’s adequate.” And the packaging led to a total yield of about 5.6×103 phages. “Try again at half the concentration and with more ligase.”
So packaging was again done, on page 127. The A protein. There were various preparations used in packaging mixes, which later on, when I was doing the Nobel Prize work, learning how to package DNA — or packaging DNA was a very important part of the operation, to be able to package phage DNA into generating a live phage. “‘A protein,’” in quotes, [00:45:00] “for packaging and cutting activity independently,” etc., etc. So with a comment on page 126 that “The A protein is most difficult to make.” Have to reisolate red plaques and so on and so for‑‑
The sonic extract, which is the other part of the packaging mix, is straightforward. And [Presol?] lysate as the sonic is used. So here were various parts used in packaging. Page 126 just lists the materials that are needed. Store it in liquid nitrogen. And the sonic extract storage also in [00:46:00] liquid nitrogen. Presol lysate instead of sonic can be used. And, “Store in liquid nitrogen.” Etc.
Fearless experiments! Going back to the mini-plasmids, the tk and G gamma. “Single colonies of the candidates were obtained. And amplify in the future.” And BamHI of minis, on Thursday, August the 2nd. The gel was overloaded. But one could still see the [00:47:00] results. These were minis, 56, 57, 114, 162, 191, etc., with 191 circled.
Are you still doing it? It’s about time to quit, I think.
Gel was overloaded. “Use less — But almost certainly, I have what I need, probably plus extra,” with arrows pointing to the products that were what one hoped to get.
Repeat of the ligation for [Bam1?] shotgun, on August the 2nd. “About 20,000 [00:48:00] phages obtained.” Going on with these, on page 135. Conclusion that “R1 plus RNase is an excellent method. Many of these products are, however, from the left side of 6.9 and several have pBR322 as a dimer. None is perfect. So screen a new set, using the 1.6 probe and using replica methods.” So. But a lot of clones were not what was wanted. Screening the various ones, with a gel there showing what they had in… More tk plus G gamma, on page 139. [00:49:00] “192 more candidates. To 37°.”
Finish them quickly.
Very soon. I’m finishi‑‑
NM:[See some?] work.
— I’ll finish this book. Yeah.
Page 143, repeating BamRI plus or minus DNA digest. Continuing in the same vein, rescreening T gamma and G gamma, on page 145. A single column is processed, according to the previous protocol — with a [00:50:00] hybridization image shown, of colonies which were positive, 247, 251, 265, etc.
A duplication hypothesis considered, on page 147, as to how, and one got from a single fetal globin gene to two globin genes, [00:51:00] with a comparison — Jerry Slightom that the sequences diverge at the end of the coding region. So considering a hypothesi‑‑ as to how the G gamma and A gamma genes arose as a pair. Which — later on turned out to be more interesting is how they communicated with each other in DNA-DNA gene conversion events. So 149, still thinking about the 6.9 piece and labeling it, etc.
And page 151, which is the next to the last page of the book, is still looking at this [00:52:00] BamRI ligation. And, “The ligation is excellent. Fragment size is excellent. And total of all current tests were used,” etc., “to give about 20,000 phages.” The last two pages of the book are end-labeling of the 6.9 G gamma piece, in order to make a probe. And that is the end of Book v.