Oliver Smithies:

[00:00:00] — little x.  Begins on January 10th, 1980, and goes on to May of the same year, starting by a transcription assay, on 30.5, an anti-gene test, you might say.  That’s what I thought it being.  An anti-gene might be used to block transcription of a normal gene.  Rather pie in the sky but nonethele‑‑ one had to dream.  So just technical elements going on the — [00:01:00] S1 nuclease treatment — of something.  Can’t see what it wa‑‑ (laughs) that was being S1‑treated.  (pause)

High-voltage denaturing gel, Friday, January 11th.  “Very promising.  —  Conclusion:  Very promising,” etc.  Back on film, to see what one obtained, with a urea, acrylamide gel.  [00:02:00] Four thousand volts was used across that gel, 40 milliamps.

Thinking about the ratio of an alpha globin precursor to an anti-alpha globin product.  It’s a dream of control, on page 9, January 11th.  Continuing this idea of anti-gene tests, on the following pages — several pages following the same idea.  Anti-Gene Test #3, January 15th.  And going on.  (laughs)  Anti-Gene [00:03:00] Test #4, page 21.  Conclusion, on Friday, January 18th, page 23, “Excellent experimental design but very little protection that is non-differential.  Remake the probes and RNA and try again, using this internally controlled design.”  So thought the experiment was good but didn’t get what I’d hope.  And repeating end-labelings, the following day.

Here’s [carfen tetranyl?] phosphatase again, on Sunday, January 20th.  End-labeling again, continuing the same general pattern, with [00:04:00] enormously hot material end-labeled.  It looks as if there was 1.1×106 counts in that preparation and 3×105 in the Pst digest.  Although the comment is that “The Pst is very poor.  But the Hinf is adequate,” on page 31.  Cutting out fragments from gels, page 33.  Thoughts and comments, page 35, etc.  The usual progression.

Now thinking about fetal mouse liver RNA, [00:05:00] with Al [Kinneberg?], on page 37.  Yield about 1.1 milligrams, on page 39, of mouse fetal liver RNA, “MFL RNA” — Book x, 39.

More anti-gene tests continuing.  Test #5.  “Results as expected.  H232 is protected.  Successful…”

(laughs)  Comments on experiments [00:06:00] in the air, with aerobatics, with a…  There was a Cessna 150 Aerobat available.  And I would try aerobatics, and successful snap rolls, too.  Meaning that I’d been flying that day.  Snap rolls are one of the more enjoyable aerobatic maneuvers, where you cause one wing of the aircraft to stall, so that it behaves as if you’d cut one wing off.  And you can roll very, very rapidly.  Hence the word “snap roll.”  They were exciting to do and enjoyable, with an airplane that was certificated to do them in and with…  These were by myself.  I know I didn’t have anybody with me.  But I had a book [00:07:00] on how to do it.

And continuing with the experiments, the next few days, in particular, continuing again — with Anti-Gene Test #6, on Monday, January 28.  Even Anti-Gene Test #6 is there, on page 37 — #7.  Anti-Gene Test #7, etc.  Continuing to do that idea.  And #8, on February Fir‑‑  [00:08:00] Typical comment of one not going right, on page 69, “Abandon!” exclamation mark.  Still very doubtful product, on that page.  Continuing these anti-gene tests.  We now are page, and 84, February 6, Anti-Gene Test #10.  As usual, Smithies seems to repeat experiments over and over again.

[00:09:00] Cutting out bands to try to get radioactive probes, on 7.5% acrylamide gel, etc, on page 89, February 8th.  Seven different radioactive things are cut out to be tested.  And these are the new probes.  (pause)  [00:10:00] Making new probes — new prep gels, and February 11th, page 97.  Still probes, probes, probes.  Again, February 12th, page 105, new probes.  “Conclusion:  The digests are complete.  Continue with carfen tetranyl phosphatase.  And cleaning up with hydroxyquinoline phenol.  There’s a…  More prep gels again.  [00:11:00] (pause)

Second cut for new probes, on February 14th, page 113.  And a protocol, and 14th — not clear where this is from — on four-base-specific reactions for sequencing end-labeled DNA.  This is talking about the Maxam-Gilbert method of [00:12:00] sequencing end-labeled DNA, the one that Nobuyo Maeda later on used very successfully.  She did many sequences with these methods.  But it was the method that we were using at that time.  It worked.

Anti‑Gene Test Gel #12, page 117, Saturday, February 16th.  As usual, Saturday and Sunday.  Makes no difference.  Saturday.  Sunday, February 17th, working too — or, as I would say, doing what I wanted to do.  Getting to Anti-Gene Test #13, page 125 and [00:13:00] following.

And on page 129, “Papers written.”  So these must have been the papers that were published with Elio, and some review articles at the same time.

With a look at the alpha sequences.  And looks as if this is a map of where the pseudogene is, on page 129, Friday, March 7th.

Mouse DNA being made.  A new clone here, 153.6, being talked about.  [00:14:00] A mouse R1 from shotgun [Bob C.?] experiment, in Charon 4A, made with [Bonnie Grebe?], and screened with an alpha‑G cDNA probe.  And three clones were obtained, 153.6, 152.18, and 152.20, with 11 kilobases and 6.4 kilobases — and hybridizing band.  Barry Whitney must have been with us at that time, too.  Because as that he was particularly a mouse person.  [00:15:00] And he says, “The two alpha are in t‑‑ are in two R1 bands, close together.  9.7 known,” and, query, “11?”  Therefore, this 11, which was Clone 153.6, is likely to be the second gene.  (pause)

But 153.6 digested in a bunch of ways.  And 4A also digested.  But, “None hybridized to the probe, [PCR 1‑alpha max?] plasmid.”  So [00:16:00] must have been somewhat of a surprise, [and experiment?].  Yet on the next page, 139, is, “Confidential.  Whitney –” Barry Whitney — “A tentative map of [litered?] mouse genomic alpha globin 2 gene.”  With a comment from Barry, “Elio, please do not reveal any of this to anyone but O.S. himself.”  So that Barry was somewhat concerned about whether he could talk about it.  But he had a map there.

Going towards the end of Book x, “A high positive O2!!” two exclamation mark, on [00:17:00] Friday, March 28th.  The idea is the mRNA degradation controls local expression.  Little did I know how true that really turned out to be, by the time we were well into 2000s, using mRNA degradation to control gene expression, with Masao Kakoki.  So this actually was a correct idea, probably, at that time, because of talking to Jeff Ross, who was very interested in RNA degradation.  But the idea is that mRNA degradation controls local expression.  For example, an increase of delta chain in thalassemia could be due to faster erythropoiesis, so that less stable delta mRNA [00:18:00] is a greater proportion of the total.  With some ideas about thalassemia.  And a comment too — oh, hypothesis too, “Pseudogenes act by diverting transcription,” away from the real thing.  With a little map of the pseudo beta gene, which produces nothing useful, and the productive delta and beta genes, on that.  So fairly good understanding, in general, of what was happening.

Ah!  Now…  I must have been teaching, at that period, because there’s a comment on, “Class has finished,” Tuesday, May [00:19:00] 20th.  And here I am, Monday, May 19th, “Origins of replication.  Control RNA transcription.”  This was a hypothesis that led me to do many experiments — that this might be a method of controlling DNA gene expression — back now in 1980.  And that sort of experiment was, as we shall see, going on very seriously at the time that gene cloning became — or gene targeting became the center of my work.  So I did publish a paper on this, which I may have [00:20:00} talked about in the pa‑‑  But I’ll just look it up again and comment on it.

(break in audio)

So is a hypothesis that I eventually published, in 1982, although I’m beginning to think it about here in 1980.  The eventual paper was, “The Control of Globin and Other Eukaryotic Genes.”  And the hypothesis is advanced that “Eukaryotes control different parts of their genomes by the selective use of origins of replication.  Replication of a gene from an origin upstream of the gene is postulated to open it for transcription, under appropriate circumstances, while replication of a gene from an origin downstream of the gene closes it.”  And the applicability of this hypothesis to the control of a human [00:21:00] beta globin cluster of genes is considered.  So that’s a beginning of what I spent quite a lot of experimental time trying to test.  As usual, I would have a hypothesis and spend a lot of time testing and usually finding the hypothesis was wrong.  But anyway, here’s the beginning of it, Monday, May 19th, page 153, in 1980, that this is the test of the origin of replication idea — with some ideas on how to address…  So that, if you transcribe in the same direction — if you replicate in the direction of the transcription, then [00:22:00] everything’s OK.  If it’s the other direction, then the gene is turned off.  And some other thoughts that possibly the origins determine also the modification patterns, variegation, which we might call epigenetic changes now, so that selection between classes of origin — of great importance in determination — or I think I meant differentiation.  But anyway, that was the beginning of that idea.

Looking for Alu repeats in the immunoglobulin clones, on page 145, with the idea, perhaps, that these controlled [00:23:00] how the genes were duplicated.  And a comment that Nan Newell, who joined me as a postdoc, has some colony.  (pause)

Various digests are being considered, on page 149, of these different clones, 165.24, 30.5, 142.7, etc., etc.  “The results show no cross-reactivity between the mouse pseudo [00:24:00] alpha and the mouse immunoglobulin gene cloned or human G gamma.  So that they were hybridized to 30.5, the pseudogene, which had been end-labeled, and then hybridized back to these clones.  And the only one which gave a result was 30.5 itself.  None of the other clone reacted, at all.  It was a very clean experiment, complete absence of hybridization, no cross-reactivity, and good hybridization to the correct sequen‑‑

So subcloning of the 3’ region of the Alu repeat, for sequencing, on page [00:25:00] 151.  And this book continues and ends on page 153, another test of digestion of these different pBR322 clones with Bam and also a test of the 7.2 fragment, etc.  And that ends Book x.  [00:25:36]


[00:00:00] This an addendum to Book x, page 43, Friday, January 25th.  I may have told this story before but I’d better say it anyway, just in case I haven’t.  But it was commenting, on the bottom of this page, the film was exposed between three o’clock and 5:30.  But in between there, I went flying and successfully achieved — snap-rolled, at 4:30 p.m.  So this was an Aerobat, a Cessna 152 Aerobat, which had stronger wings, so that it would stand aerobatic‑‑  And in January in Wisconsin, with the colder temperature, it was, oh, [00:01:00] an enjoyable airplane to fly, because it had lots of power, with it being cold.  But the snap rolls were being executed by myself, with the help of an instruction book that I used to carry with me, written by a person whose name I don’t immediately remember but it’ll come back to me.

And the story of this is that, sometime later, I went to, in my airplane, at that time a Cessna 172, to visit a friend in West Virginia.  And the weather was rather poor.  But my friend and collaborator, whom we will come across in due course…  [00:02:00] Damn.  (break in audio)  Yes, where I’d been invited, with Nobuyo, to visit Walter Nance, in West Virginia, at a place that he — I don’t know whether is his home or a cottage.  I’ve forgotten that.  But anyway, part of the scheme was to visit with a pilot friend.  And we got talking about various things.  And I said that I’d been carrying out various aerobatic maneuvers, using a book that I’d found a great deal of help.  And he got a bigger and bigger grin on his face and [00:03:00] then eventually said, “Yes, and I wrote that book.”  So it was an enjoyable occasion.  So I was using his book at that time.  And I’ll find his name, momentarily.  (break in audio)  His name was Bill Keshner.

And he was famous for this book and for his training of people in spin recoveries, that many people were quite — even now, frightened of spinning an aircraft.  And if the aircraft is certificated for spins, then it’s a part of training which it’s wise to carry out.  Although even that is debated.  Because some of that has been taken out of the requirements of flight training, [00:04:00] because too many accidents occurred during the training.  So it — a debatable item.  Although I used to enjoy teaching my fighter pilots to spin, in the Blanik, because it was a very safe aircraft to spin.