Oliver Smithies:

[00:00:00] So this is Book w, which begins on August 9th, 1979, and goes through to January of 1980, beginning as a continuation of the work, trying to have the thymidine kinase gene driving G-gamma in order to understand the control of the gene, or perhaps  it was the other way around.  Anyway, the idea being to learn the features of control of the mammalian genes G gamma, the mammalian gene G gamma.

So first page is minis of new candidates. [00:01:00] And this continues for several pages.

And labeling of a 6.5kb G gamma fragment to act as a probe on page 3, August 10th, Friday, and analyzing the results on Monday, August 13th, [proceeding?] things.  The TK gene is next to G gamma, two particular clones, one called 353, and the other called 251.

And mapping the sites on the 6.9 gamma piece, [00:02:00] and further work of that type on Tuesday, August 14th, page 13.

And Jerry Slighton’s data being looked at on page 15, and what we might expect in the following experiment.

And here again on Tuesday, August 14th, page 17, working with these two TK, G gamma, G gamma, TK G gamma clones, 251 and 353.

Continuing on several pages, with a comment on the data analysis of a gel on page 21, “Best bet is to sequence [00:03:00] to the right from the left end of the 2990 piece.”  Quite nice gels on the left, but no labels, so it’s questionable if I ran them.

Here is some more discussion of what’s going on on page 23.  Both alpha globin genes that is, are likely retained in the screen for alphas, and it would be useful to have both gammas, and delta, and beta on single plasmids, (inaudible) the foresight of something that turned out to be really quite interesting later on, when we got the G gamma and A gamma genes on single fragments.  So, thinking about them being useful, which later proved [00:04:00] to be the case.

On page 25, we are talking about Friday, August 17th, through Sunday, August 19th, characterizing two pBR322 clones which we received from Tom Maniatis who, as usual, is very generous with everything, so we got from him a beta, and a delta clones, processed as previously on page 25.  Good separation, but poor yields.

Continuing with the characterization of earlier clones on the following page.  On page 29, [00:05:00] for example, the second gradients are rather diffuse, et cetera, et cetera.  Yield here, one case was 524μg, and the other was 435.  And these were sent to Raju Kucherlapati, his assembly into the test vehicles that we were going to use.  I will, no doubt, find that paper in due course, because we did publish it.

Continuing on page 31, re-cloning the TK with G gamma and A gamma, and [00:06:00] G gamma + A gamma, re-clone, on page 31.  So, this work is continuing with these two plasmids.

“Minis on Maniatis’ H alpha G2,” doesn’t say specifically what that is.  It was a probe, or clone obtained from Tom Maniatis.

Continuing in the same vein, over the next few pages.  So, on Thursday, August 30th, back from Princeton.  Must have [00:07:00] given a seminar there, minis from Maniatis’ alpha clone were processed.

And the TK beta plasmid now being considered on page 49, referring back to page 9, where there were various possibilities being considered for beta and TK, talking about how it might be made, beta TK can be made as a dimer, going back to page 9, briefly.  And unstable in one orientation, et cetera.  And so this was a continuation of the ideas started on page 9, and now on page 49. [00:08:00]

Tried to digest in a simple experiment, but conclusion, “Overshot badly; do a proper titration.”  So a titrating of the [HB1S?] for a single cut on the following page, to get a single cut rather than multiple cuts.

Continuing on Tuesday, September 4th, page 53.  Still looking to get that, with a conclusion, “Almost ideal, a [trace high?] digestion.  Preparation as usual.  Excellent product.”  So the single cut of that plasmid was made all right.

Considering making a probe for beta globin. [00:09:00] Screen for beta colonies using delta Pst fragment, since beta overlaps pBR322, et cetera, technical problem.

And ligation now of the TK to the beta globin piece on page 57.  “Ligation excellent,” et cetera.  And remaking starting again the single cut on pBR322 TK on September 6th.

Now trying to get the sequence between delta and beta, “To test whether delta and beta are co-transcribed. [00:10:00] It will be useful to have a probe from between them.  A suitable probe is,” there is a map of what might be a suitable probe.  And a comment, (inaudible) has something from which that could be made.

So, Bam cutting pBR322, imagine having a whole page for that.  BamRI cut of pBR322 still going on on page 67, something that will be very routine, but still [00:11:00] worth having a page in a notebook at that time.  Usual business of ligations, the Stockmeyer concentrations were always (inaudible), somewhat unnecessary probably, on page 69, with some doubtfulness about whether it was correct or not.  But the ligation, one ligation looked excellent, and one looked reasonable.  That’s Tuesday, September 11.

And can make the same set of clones for the fetal genes, as commented on page 70 in getting out the transformation to get 950bp beta-IVS [00:12:00] fragment, and 700bp delta beta, between delta and beta fragment, that had been talked earlier.  This type of work continuing over the next several pages.

IVS tests and ligations on page 77, Saturday, September 15th.  Conclusion: “Good preparations.”  So everything was proceeding in regular sequence, if somewhat slowly.

Now, a probe now for the G gamma + A gamma, TK pBR322 being considered on September 17th, page 81. [00:13:00] Need to have the point as 58kb pair R1 piece on 3’ end of A gamma as a probe for the complete G gamma + A gamma subclone.  And the number of probes up for test now on page 83.

And, on this note, this was on September 17th, and October 24th, quite a gap on the next page, “Back from a vacation, a meeting in Paris with [1H2RG?],” that’s an airplane of that type that  [00:14:00] I had to Spain, and back from London, et cetera.  So, a good vacation flying.  Guy Fawkes Day, a comment on November 5th.  H alpha G2, and so on, “Poor amplifications that day.”  The characterization of the different pieces obtained. [00:15:00]

Talking about stringency of hybridization on page 97, to decrease nonspecific cross-reaction, et cetera.  Need to [watch/wash?] to greater stringency.

And repeating genomic Southerns on page 99, a previous example being [00:16:00] on page 92.  But no result showing immediately on the Friday experiment, November 23rd.

Photographs of the stained gel on page 102.  And there, we come to the autoradiograph result on page 104, Sunday, November 25th, where the Southern blots are shown, the conclusion being that, “HhaI doesn’t cut well due to [00:17:00] methylated cytosine.”  But they’re not bad Southern blots nonetheless.  One with the 2.9 probe, and one with the cDNA probe.  Results are very similar.

So a new clone, 164.6 being checked on.  Jerry Slighton has tested that clone, and concluded that it has G gamma and the A gamma [00:18:00] gene on one piece, which, in the end, was a very useful piece of DNA to have, and we learned a lot from it.

Many tests on 165.24, and 164.6, and 51.1, et cetera, on page 111, with the results on the left-hand side, “Very satisfying gel.  1% agarose, Helling’s x4, 3.8V/cm,” [00:19:00] et cetera.  “A good gel.”  And the interpretation of the results on page 113.

Considering using a different enzyme instead of HhaI Sunday, December 2nd.  And, the execution of that on the following page, with, again, a rather beautiful gel, and quite good radioactive blot, and with the conclusion that, “TaqI worked fine.  But, the [00:20:00] [two/too?] little, or [two/too?] short RI fragments,” that was TaqI and TaqI + EcoRI.

And now, a summary of the differences between the various clones with a rather formal diagram on Sunday, December 30th, with two other comments, the paper was written, which we’ll have a look at in due course.  This is Sunday, December 30th, and I passed the commercial pilot exam, so I’m now qualified as a commercial pilot.  That’s not airline transport, but commercial meaning, at least you can fly for hire, not that I ever wanted to.  But it’s a marker of one’s progress. [00:21:00] So here is the summary of the difference between the adult alpha globin clone and 30.5, with various thoughts on it.

Some work on cloning, or preparing cDNA from mouse globin on December 31st, page 125, and a protocol on the opposite page for making cDNA, just technical things.

30.5/700 [00:22:00] repeat on following page, I will eventually remember what “30.5” is, but for the moment, I don’t remember what it was, but it was a number that I’m still familiar with.

A different strategy being thought up for getting specific hybridizations.  Al [Kenburg?] suggested using kinase for end labeling, and S1 endonuclease to [see?] protection, using this idea, following sites would look good as (inaudible).

So 30.5 being tested further on Friday, January 4.  With, in this case, a 7.5% [00:23:00] acrylamide gel.  Gels are really quite long gels.  Here’s an image of one on page 134.  The gel was more than 20cm long.  7.5% acrylamide, with [half X?], Tris-borate-EDTA gel, and 25% glycerol.  And the conclusion that, “The digests are perfect.  BstEII, HinfI,” et cetera, on page 135.

Calf intestinal phosphatase being prepared [00:24:00] or used on page 135.  Get rid of any end label, any phosphates to enable end labeling of DNA.

Another possible labeling scheme being talked about on page 140, and end labeling going on, 32p-end labeling on Monday, January 7th, with an autoradiograph showing very heavy labeling.  I’m trying to see there how much [00:25:00] how many counts were used, about 250 microcuries of gamma-ATP, 3,000 microcuries/mmol is enough to label 10μg, so quite a hefty amount of gamma-labeled ATP used in this experiment.

And continuing.  Now we get to some sequences, with the complete sequence of 30.5 on page 149, and I have to look up a little bit more to find out what that is.  So I’ll pause for a minute.

So this book ends rather nicely with the [00:26:00] complete sequence, or almost complete, it says complete sequence of the clone 30.5, which turned out to be a pseudogene, a mouse alpha globin-related pseudogene, lacking intervening sequences, as we published; Elio Vanin, and Greg Goldberg, and Phil Tucker and I published later in July of 1980.  So this was finding that we had spent rather a lot of time on something that was interesting, but not quite what we had wanted to get, but still had a bearing on the way evolution works.  This is because it was a copy, a cDNA copy presumably from RNA made during evolution, [00:27:00] because it lacked intravenous sequences.  It couldn’t code for a functional globin polypeptide because of frameshift, and commenting on, “The widespread occurrence of globin pseudogenes in other species suggest that they are not dead genes, but may be important in controlling globin expression.”  That did not turn out to be true, but they’re interesting nonetheless.  And that was the end of Book w.