Oliver Smithies:
[00:00:00] Yes. We’re now starting Book k, lower-case K, December 30th, 1974. Goes out to June of 1975. The beginning of this book is still continuing to work on different gel procedures, with a view, I recollect, to being able to sequence radioactive material available only in small quantity. So the first recognizable result is on pages 2 and 3, comparing a separation on an acrylamide gel, [00:01:00] polyacrylamide gel, that Byron Ballou had made, with the separation in a starch gel, of various iodinated preparations of beta‑2 microglobulin. And the gel staining is there and the autoradiograph. Quite clearly demonstrated.And this is followed, on Monday, December 30th, page 4 and 5, by an experiment in which Byron had 14C-labeled mouse cells and ran on his gel, with polyacrylamide. And autoradiograph obtained. Beginning to test having anti-beta‑2 microglobulin on [00:02:00] Sepharose beads, on the following page, “Thursday, January 2nd, 1975!” exclamation mark, with some gels, again — looking, in this case, with a gel that became later a great favorite, a chlorohydrate gel — aluminum lactate chlorohydrate gel, which Byron had developed and which we published on, but sometime. The separations really were quite remarkably nice on page 12, two gels, one better than the other. But both are very good. And the autoradiographs [00:03:00] were fine.
Continuing then to look at the starch gels, on the next page, with, in this case, just beta‑2‑M labeled. Antibody B experiment repeated, on page 17. Usual way of repeating things many times. Slightly variable gels, horizontal versus vertical, a chlorohydrate gel. Which, the vertical one looks considerably nicer.
Specificity of the beads being tested, page 19. And a [00:04:00] bead capacity test on page 23 and so for– A column elution was looked at, on page 24 and 25, with a comment that this is the supernatant and the beads and the eluate from the beads, two gels, with a comment that “It’s clear that the beads have a tremendous capacity and increase in specificity [of its record?], as the volume decreases. No limits seen in these tests.” So we were quite encouraged. And going on in the same vein, compared Byron’s 14C experiments with the GM‑17 and GM‑17 in [00:05:00] 60-dishes and in [roller?] bottles. Different experiments.
Bulk preparation of these samples, on page 29, January 24th, continuing this general way of the — labeling beta‑2 microglobulin in bulk, in this case with [3×50?] microcuries of 14C label. This was material that was being given to Dave McKean for his radioactive sequencing. [00:06:00] With a comment on page 30 that “About 11,000 counts were ma‑‑ total, enough only for one-day run on the sequenator.” So we were using the sequenator to do radioactive sequencing. And continuing in the same vein, with more column, on January 30th. And repeat of absorption, on February 3rd. Columns, on February 5th and so forth, all continuing the same general pattern.
On February [00:07:00] 5th, Wednesday, we decided to label 200 microcuries of 35S methionine, [per the?] — and test each cultured strain from Kucherlapati. Now what has Kucherlapati got to do with this? Well, that isn’t immediately clear from the notebook. But it is clear, if we look at the paper that eventually came out, in 1976, which was the first publication I had with Raju Kucherlapati and was, in fact, the beginning of a long and enjoyable collaboration, that extended to the time of gene targeting — so in 1976, so later than the book. But this is a prelude [00:08:00] to this work. Raju and all the others [over?] — assigned the gene for beta‑2 macroglobulin to human chromosome 15. And this was done by taking hybrid cell lines with different chromosomes in them, that Raju had developed in Frank Ruddle’s lab. Frank Ruddle was an author in the paper, of cour– And then anti-beta‑2 microglobulin serum was coupled to beads to make the immunoabsorbent that we’ve been seeing in the notebook. And parental and hybrid cells are grown in medium containing 35S methionine, etc. And [00:09:00] the beads were eluted and the supernatant and the eluted material was looked at. And the gels were the chlorohydrate gels that are being used in this part of the notebook. So the chlorohydrate, the immunoabsorbent, and the ability of chlorohydrate gel to separate mouse from human beta‑2‑M was used to assign the locus for beta‑2 microglobulin to human chromosome 15. That was the best that could be done, in those days, was to put it to a chromosome, not a specific locu‑‑ on the chromosome. So here we’re [00:10:00] continuing this work in this book.
Inhibition tests with Triton extracts, on Friday, February 14th, with a note that this is Valentine’s Day. Human-mouse linkage absorption, page 17 — I mean, 47. And continuing with that book — progressive. Byron labeled cells with cysteine and methionine, 35S. Anyway — so that’s not quite correct. Labeled with 35S methionine, about 1,500 microcuries. Must have used a fair amount of lead. [00:11:00] One-point-five millicuries. (long pause) Rather spectacular set of images, on page 62. Every page thereafter seems to have a label, [00:12:00] ju‑‑
A little return to nuclei isolation, on Wednesday, April 16th. “Get meiotic histone trials for Ed Sheldon. This was not one my experiments. It was to help someone else. [Oh, for?] meiosis divisions I, II and [test trans-mixture?], etc. So isolating nuclei. “Unfortunately, thawed overnight.” [00:13:00] Going on, in the next few pages, with that experiment. Typical comments, on the way. “Suspension easy. Pellet was almost solid.”
And on Wednesday, April 27th — April 23rd, rather, page 77, following a paper of which a copy is attached, [00:14:00] by Hewish and Burgoyne, “Isolation of Chromatin DNA at Regularly Spaced Sites by Nuclear Deoxyribonuclea‑‑” So this was then on April 23rd, attempt at chromatin subunit isolation, for use in attempting compositions and identities and separations for Liz Freedlander’s experiments. Oh, what Liz was trying to do was follow histone segregation with DNA. So here we are, Thursday, April [00:15:00] 24th, chromatin subunits, continued. Different buffers for isolating the nuclei, several pages of it. Here we are, on page 97, still doing…
And then were using the chlorohydrate aluminum lactate gel to test the histones, on page 99, with a comment that “[Caroline?] repeated the experiment. Still poor. Therefore, try in Byron’s apparatus.”
[00:16:00] Here is a different approach being considered, on Monday, June the 2nd. “Glass beads/Sequemat, initial trial.” That’s a different type of sequenator. The aim, to get high-sensitivity, [cold?] sequencing working. And, “Plan tests,” etc., “in the regular sequenator, plus or minus carrier,” and so on. “Beta‑2‑M looks suitable for both tests with 125-iodinated material. Can use lysine and cysteine coupling methods,” etc., “for Sequemat.” The Sequemat had the protein coupled to beads. Tha– [00:17:00] So that was work being done for the following few pages, again, 105 and 107. One-oh-nine, coupling to the glass.(laughs) Comment on page 115 that “Joe sequenced OK [Dunn and Parrow?]. But the sequence was very poor.” (pause) Trying to use material [00:18:00] that had been coupled and then cut at methionines, with cyanogen bromide, on page 117. A check of glass without DITC, whatever DITC is — with a comment of about 180,000 counts per minute and the results show the sequence is detectable even without coupling, at [00:19:00] low level. Repeat of lactoperoxidase labeling with 125I. Page 123, different methods of — or different attempts to label. Sequenator experiment, on 125, commented. About 113,000 counts per minute, for use in the sequencing. Sequemat, on page 127. Sequemat procedure is specifically [00:20:00] listed on page 128, check of the solvents, etc., etc. — and my notes on how to use the machine.
And this is an [experiment?] that Dave McKean had developed. And there’s a reprint from Biochemical Journal, 1974, attached to page 130, which is, “The Use of Radiolabelled T1 Ribonuclease to Monitor the Efficiency of Edman Degradations” — Dave McKean and George Smith, both of whom were my postdocs in the Laboratory of Genetics at the University of Wisconsin. So this was in [00:21:00] February of ’74, almost a year earlier than we are in the book — well, almost six months earlier.
More radiolabeling going on. With a result, on June 16, saying that “The autorads show that the crude beta‑2‑M contains many labeled components in addition to beta‑2‑M.” [00:22:00] And also a comment that “The highly purified beta‑2‑M, from, for example S‑3, page 39, K135I, shows very little impurities.” So we were able to purify it but not all preparations were pure. Dave McKean sequencing, on page 104 — sorry — on page 140, sequenator run [1054?] — with three peaks.
[00:23:00] Now I comment, on this page 141, on the sequenator efficiency. It comments on the fact that alpha… First of all, the yield of T1 was 60.5%. Alpha was 94% and beta about 2%. The terms alpha and beta correspond to the efficiency of cleavage in the sequenator. Ninety-four percent of an amino acid would appear at the correct position. But then beta was a correction factor for what was carried over to the next [00:24:00] cycle, because it hadn’t cleaved. So alpha and beta were parameters which I had developed, with the help of Bob Goodfliesch, in working out the programs for efficient sequencing, recovering good results from the sequenator, even though the machine was not perfect. (pause)A note on page 147, “DeMar’s strain 7223…” That’s Bob DeMar, who was in [00:25:00] the neighboring lab, and at a time when he and I were talking together. We had some later unfortunate disagreements. But at this point, this is a strain from him. And Brenda has some other strains. And that’s Brenda Cairn — some other… These were different strains of cell lines, I presume.
So June 19th is a quite nice page, page 149, sequenator runs, at 1057, 1055, and 1058. “Results confirm good sequence with 14C-labeled T1 plus 1.3 [00:26:00] milligrams of Braunitzer. Alpha is 92%, beta 2% to 2.5%. Yield at 2 was 44%,” etc., etc. More beta‑2 for sequencing, as we get to the end of this book. Which, the last page has a chlorohydra‑‑ has an attached typewritten man‑‑ instructions for the chlorohydrate gel sys– “The following systems have proved useful for separations of membrane proteins.” So one is an aluminum lactate gel mixture. Etc. And ammonium formate gel, as well. And [00:27:00] two pages of instruction. End Book k. [00:27:06]