Oliver Smithies:
[00:00:00] On Book g, still continuing, starting November 25th, 1973, and running through February of 1974. So again, not a very long period of time.Starting on Sunday, November 25th, I haven’t made any comment, by the way, that all these images appear to have been taken on Polaroid film, at least some were. This is quite obvious on the gel image on, facing page 1. [00:01:00] 4X Helling’s, 0.5% [C-chem?], 2V/cm, run overnight. And even by what I would know now, the electrophoresis is fairly good, especially as one might expect of the digest, where the DNA are smaller, with a comment that, a DV-653 appears a lot faster than DV-1, but doesn’t check with the run on F-48, so trying to use the gel to determine what’s going on, much faster than experiments with the electron microscope, but can be used to guide [00:02:00] the electron microscope study.
RI digest from Fred Blattner, lambda dv tetramer, optical density 1.2, digested by Fred, made by Randy; I don’t remember who Randy was. I could probably find out, from Fred Blattner’s lab, one digest in big capsule, and then on the following page 5, Monday, the 26th, these digests that, looking at it, the dv F-11/30 digest, very clear multiple [00:03:00] bands of a typical phage digest, but lambda dv F-144 digest was rather poor. And then a whole bunch of RI digests of control lambda dv, 453, shows no effect of the digest, which perhaps means there was no RI site; let’s see what I conclude. Fred suggests that diluting the enzyme might have killed it, or the temperature, and then tested RI over 5, plus the control briefly, and ran on a 10V/cm gel and came to the conclusion that the enzyme is dead.
So repeated on the following page. [00:04:00] Helling’s 4x, and dv digests, and lambda dv digests, and no digestion. But the gels are informative.
Going back to talk about the electron micrographs, and so, electron micrographs of F and S, from F-140, Book f 140, show all bands have the same length, but S is mainly linear. ?F is circle, ?[faint?] pre-S [00:05:00] with one nick, and S with a double-stranded break, was the thought.
Continuing this type of thought, for example, on page 10, with a comment that if there’s [a whole surface?] circle, that the circle of double-stranded DNA versus one nick, or versus two double-stranded break is confirmed, we can recheck with pronase, etc., etc., with some solid [00:06:00] Orange G dye, pKa 13, to look at what was happening to the pH.
Learning to digest in different ways, in the following page. RI digests of 653 on November 30th, page 17, with diagrams of what I interpreted the products to be. Conclusion that [00:07:00] the enzyme is stopped by heating at — this is RI digest — the enzyme is heating to 70 degrees, and Helling’s by [4R?] buffer with 0.5% agarose gel, will resolve well. What became again, the standard protocol for making gel.
RI digest at 0 degrees Centigrade on Saturday [00:08:00] December 1st, and with thoughts of varying the sodium chloride concentration to shift the reaction rates. Of course, it would have been better to think about magnesium, but I don’t know that I was sophisticated enough to know that.
Rather poor pair; one gel poor, one gel good, on page 20. The bottom gel is really quite poor, but the top gel is quite a nice gel. I’m looking at RI, and HI plus HindIII now, called HindIII, it would be HindIII, cuts of lambda dv 653. [00:09:00] Where RI does not cut, but RI plus HindIII appears to cut; I’m not quite sure what the conclusion is, but anyway. The comment says, “Looked as if the samples were mixed, it suggests that HindIII old was really RI plus HindIII, and UV, etc., and repeat, some problem about labeling and whether the samples are correctly labeled. So the repeat is set up [00:10:00] on page 25 of lambda dv 653, with old HindIII, new HindIII, etc., and Bernie’s RI gel had a problem, accident, cooling fail. Even so, clear that HindIII plus RI is equivalent to HindIII, equivalent to RI to a first approximation. Only the old HindIII is different, confirms an earlier gel, not at all obvious what is being meant there, but they’re trying to solve the digestion problem.
And continuing these tests on page 27. [00:11:00] And the beginning of plotting migration on a logarithmic scale, on page 26. G-26 gel, phage, and plotting the migration of samples on the gel. First plot of that type that I have seen in the notebooks at this point. [00:12:00]
Going on with digestion of various preparations, and now beginning to have a map of what to expect with different preparations. These are E. coli DNA presumably, and a map on page 32, and showing KH-54 DNA, and beginning to see where the enzymes cut in the various preparations, so learning to look at maps of DNA. [00:13:00]
HindIII cuts on page 33. Now again on page 34, a nice plot of the G-34 gel, where size migration are on a straight line, with a logarithmic plot. Quite a pretty plot.
Page 35 was RI and HindIII rechecks. So continuing with these double digests, single and double RI and HindIII. [00:14:00]
With a realization on page 39 that the order of adding the enzymes and plus or minus inactivation could be important. And with the conclusion that it’s therefore safe to use HindIII and inactivate, before then following with EcoRI digest.
Time series with HindIII on page 41, and with the gels on page 40. [00:15:00] Comment that, “Disappointing. The kinetics are very dubious. Looks as if all the enzymes are dying very rapidly, and are therefore all-or-none digests, that there isn’t a time effect. You get an initial time, and then nothing further happens,” not yet knowing how to digest DNA.
Stability of HindIII, of Hin and RI stability tests on page 45. The results confirm the instability of RI, which has not improved by mercaptoethanol. No evidence that HindIII is unstable. [00:16:00]
Stoichiometry tests of HindIII on 47, to test the effect of different amounts of enzyme. It’s quite a nice gel, 1% Helling’s, 10V/cm, and a very clear result of, this in the presence of ATP HindIII, that the digests dependent on the amount of enzyme added, and repeat exactly, but using five minutes for all of them. but the result is a very definite demonstration that you have to digest completely, [00:17:00] and have enough enzyme.
Some tests of HindIII and EII colicin. Colicins behaves somewhat like HindIII, therefore talked to [Namuro?]. He suggested that EII would be the best candidate of the colicins. And, the conclusion being that this preparation of EII is a powerful nuclease [00:18:00] at this high concentration. Test lower, just digested completely, and gained no specific band. EII further tests, slight residual activity at 1/10, but nothing below. No sign of a useful enzyme.
And conclusions of a HindIII digestion on page 55. Looking for sticky ends of pgal8 on page 57. [00:19:00] Some DNA from [Rading?], very promising, looks as if B-189 [Rading?] is clean, except for a cut between P and Q, looking at the maps. This is a lambda lysogen, I imagine.
Good experiment on page 61, Phi-[AT/80?], [Cigna/Signa?], and B-519 tests [00:20:00], looking at the products, and plotting the gels. The result is [Cigna/Signa?] and [Phi-80?] do not have the thick 13.5% Rad band or the 99.5% C-72 band; therefore something is wrong.
So, continuing with the analysis of gels and bands of HindIII data on page 62 and 63, quite good gel. And, attempt the scheme on page 65, showing all of the [00:21:00] maps of all of the lambda lysogens, and what I thought they should be. And then, comparing digests on the gel. So, mapping these bacteriophages on the bacterial lysogens has begun to take over [00:22:00] in these experiments, the ultimate aim of which is getting lost. I seem to have been hooked by gel digests, and mapping. Not an uncommon problem of mine to get hooked on a different type of experiment while attempting to solve a primary problem. Though here again, dv squared Gal-120 tests, [00:23:00] perhaps getting a little closer, but the gel is rather a mess.
Bulk dv Gal-120 on page 79, Monday, December 31st. The comment, “It looks very promising, [00:24:00] and prepare individual bands for tests.” Worrying about Gal-120 not being cut by HindIII scheme if it wasn’t, on page 81, New Years Day.
New buffers being made for the digests [00:25:00] on page 85, worrying a little bit about them getting into trouble, and now not enough of the restriction enzymes.
New bacteriophages on Friday, the 4th. [00:26:00] Continuing to test digests, Saturday January 5th, page 95, in big, black magic marker, type of thing, Fred [solved it?], keep the 9% cut as one, and use the left cut, that’s the second, showing an example of what might be done in order to get, on the left-hand page, the replication bubble. [00:27:00]
Protease K tests, and new HindIII. Daniel [Stafford?], Department of Zoology, UNC, whom I later came to know much better, Stafford at UNC. He uses a proteinase K at 60 degrees, in place of protein, pronase, [00:28:00] and gets low nick rate. As I’ve said, of course, that’s a problem if the enzymes have nucleases in them, if the proteolytic enzymes have nucleases in them, you can’t get a good product. And the conclusion from this is that proteinase K is equivalent to pronase at 10X concentration, so use it. But no signs of nuclease in either of them, so both of them were good the pronase and the proteinase K, but proteinase K is better.
Marshall Edgell and Clyde Hutchison, these are guys in North Carolina whom I’ve come to know much better at a later state. First mention I have ever seen of people from North Carolina a good notice. So I’ll [00:29:00] look at this page again. Marshall Edgell and Clyde Hutchison gave me new mutant strain, Haemophilus influenzae, and some of its only Hind, and probably same as HindIII, etc., etc., a little bit (inaudible), but the conclusion is, etc., as mentioned, about the pronases, but here we are. Marshall Edgell, who was a lifelong friend now, and Clyde Hutchison, who is no longer alive, but was for a long time a friend also at Chapel Hill.
A note here is of Daniel Stafford, and it’s actually [00:30:00] Darrel Stafford, but I didn’t know that name as a first name at that time, so I wrote down “Daniel Stafford,” but all of these people became later much influential in my going to Chapel Hill with Nobuyo Maeda, and their inviting me to come following her. So, Marshall Edgell, Clyde Hutchison, and Darrel Stafford are introduced to me on Saturday, on this introduction in the beginning of 1974. It’s going to be [00:31:00] 15 years before I pursue that contact. These two pages must be considered quite precious, not because of the science, but because of the scientists on those pages, who later, as I said, were very important in my history.
So continuing on that recipe on the next page from Bill Middleton on restriction endonucleases from Haemophilus, (inaudible) the Hin enzymes. We’re going on with phenol extracts, page 105 [00:32:00] January 15th, bulk preparations of F1GAL lambda, and digesting it with HindIII for electron microscopy on page 109. The gels have the result. The results show no further RI action on second test, or that it should have been heated to melt [overlaps?] etc., etc., testing RI digests of the different materials. [00:33:00]
The next page, page 113, is concerned with some beta-2 microglobulin experiments again, from [a rice field?], one had 36mg, and one had 43mg. This was cutting beta-2 microglobulin with cyanogen bromide to cut at the methionines on the following page, 115, and also cutting with papain on [00:34:00] page 117. So why I was back with beta-2 microglobulin at that point, I’m not really quite sure.
Sunday, January 20th, trying to digest lambda 72 with RI and with a comment that the results show that the RI is nearly dead, only a little left. There’s a new sample. [00:35:00] Repeating the digests, trying digests with or without [00:36:00] W2, without L1, whatever “L1” was.
Repeating lambda pgal8 for electron microscope on Thursday, January 24th, so the aim of electron microscopy still continuing, with a comment on page 127, “Good W2 with L1 is OK.” [00:37:00]
Trying a different enzyme, Serratia enzyme from Bernie. A quite good gel, and reasonable blot versus size. [00:38:00]
Continuing to map these deletions on possibility of deletions on page 131 of W2, L1, cut HindIII, or Hin, [phenolate?] it, and back to dialysis. Comparing W2 with HindIII again. [00:39:00]
A whole [raft?] of instructions on page 136, from Dolores [Stephenson?], how to spread the DNA, very detailed, typewritten instructions. [00:40:00]
So continuing with preparing HindIII for the electron micrograph, HindIII [lysogens?]. The plan was on page 138 to take 1W first, and cold, etc., and seed on Thursday, feed on Friday, block on Sunday, unblock on Monday, and label, change to cold, re-block or harvest on Monday, and release Tuesday, harvest or re-block on Tuesday evening, so scheme [00:41:00] with a different sample numbers.
These are experiments [00:42:00], it’s not clear from here what cells are being used here in this experiment. But there are some type of eukaryotic cell, probably, because the Pen strep was being used, and [calf serum?], [00:43:00] and Coulter counter of these cells. And the cells were being fed, etc..
Complete change of direction in these, because here was harvesting chromosomes on page 145, and the colchicine used to [00:44:00] block the cells in mitosis. And, this is continuing. The following pages are, have nothing. That’s the end of the book, but there are a whole bunch of W2, L1 control, etc., images, that may make some sense if I look at them.
I guess, the book ends with about 10 images, electron micrographs that are very dark, and very difficult to see [00:45:00] that there is DNA on them, that these are W2, L1 control, W2, L1, HindIII, diluted 1/27, etc., diluted 1/9, and so on and so forth, control diluted 1/9, and if one looks very carefully, it’s possible to see that there’s a large amount of DNA on these, but the images are really very difficult to see, and there is no attempt to interpret them at the end of this book, but there was a large amount of DNA on these grids. And so that ends Book g.