Oliver Smithies:
[00:00:00] So here we are on Book h, February, 1974, still in Wisconsin, going on up to August of the same year. So it starts off with Liz’s tryptophan label experiment. This is the beginning of Book h, and here, the first experiments are in relation to experiments which Liz Freedlender was doing to test the segregation of [00:01:00] histones with DNA. I had had at some stage in that period a hypothesis that I was, as I explained before, that histones, and the orientation of histones in chromatin might be important in conveying information, and Liz had been interested in looking at the distribution of chromatin-associated proteins, and so, a paper that was eventually published by her and Lorne Taichmann, and myself, in 77 [00:02:00] was concerned with this, and nonrandom distribution of chromosomal proteins during cell replication. But these, then back in 1974, or some of the experiments related to that paper, and to the idea that I had in my head, and so, there’s a schedule there set up by Liz, using Chinese hamster over [itself?], presumably, though it doesn’t actually say that; just said, as adding cells. So initially, [00:03:00] with tritiated thymidine, and tritiated tryptophan in the other experiments. And then, colcimid to get mitosis, and looking at slides, and so forth. So this is trying to look at the chromosomes from cells labeled in different ways.For example, on page 8, from Lorne, shake after metaphase, and decant, etc., and pellet, and combine; remove supernatant, etc., etc. [00:04:00]
So the first metaphase chromosomes are being considered on page 11. And then, on page 13, isolation of second metaphase chromosomes. On Wednesday, spread about 20 slides, and labeled them S13. Third and fourth metaphase on page 17, [00:05:00] with some comments on Thursday, February 21st, four hour, +4t chromosome, label 4+ moderate quality, 3 rather poor, 3t+5t virtually useless, etc., and 5 adequate.
Notes of Lorne on what was happening on the following page, and an inset, what had obviously been a Cyclostyle reproduction of [00:06:00] typewritten instructions on autoradiography treatment with radioactive thymidine, etc., dipping methods and equipment for the autoradiography, interesting to see some Cyclostyle material, because that was the basis of Ed Southern’s development of Southern blots, which because of his knowledge of this method of reproducing copies of material, quite good quality reproduction, all in blue. Friday, February 22nd, that’s page 23.
Continuing, a dip of the [00:07:00] slides, and so on. They were dipped into the emulsion and left for some long period of time. Third and fourth chromosomes, Friday, the 22nd, etc. Unfortunately, on the opposite page, page 26, “Check the final product. Extremely poor; hardly any two-arms.” I needed to see the two arms of the chromosome.
Charlie Heidelberger comment on 10mM tryptophan general labeled, [00:08:00] “no W going to bases directly, might exchange,” etc. Some comments, “Things went OK. Agrees that relief of block for one hour should secure it,” whatever that means. I’m talking to Charlie Heidelberger.
A lysine label on Monday, February 24th. Talking about blocking DNA replication with thymidine. Decided thymidine is too unsafe for base synthesis, and so we’ll relieve the block for about one hour before labeling. [00:09:00] A rather complicated set of experiments.
DNA in nuclei on February 25th. Continuing, for example, on Tuesday, February 26th, followed by Thursday, 8:30 a.m. harvested, very low yield indeed. Probably they were stationary, since I forgot to use half seed level, etc. [00:10:00] Attempt to get good chromosome spreads.
On Friday, March 1st, “Liz thinks the developing failed. Developed #2 H1 essentials, as still looks bad, but continue and redip.”
Results, Saturday of H1 experiment, six-day exposure. [00:11:00] “Definite asymmetry, and some half label, not as good as seven,” etc. “Number 89, no chromosomes.”
H1 Friday March 18th, 13 days, so not easy experiments.
A new tryptophan experiment being considered on Monday, March 4th. Repeat once again, but concentrate on second and third metaphase. [00:12:00]
And so, often we are talking about days later than the experiment was done. Wednesday March 6th, “Developed a five-day exposure of the lysine label experiment, second metaphase. Extremely heavy and dirty preparation,” etc. “Conclusion: wait to ten days for the next development and treat with,” this, that, and the other.
This [00:13:00] type of experiment just being continued. Chromosome from H49 experiment, or H57, on Saturday, March 9th, etc. Some “(few usable in both preparations)” a spread. The difference between whether a tube should be a plastic tube or a glass tube, being worried about on page 60. The constant [00:14:00] interest of mine on what sticks to various plastics, or glass, or in some cases, petroleum jelly, petrolatum, used to make non-sticky surfaces. I’m constantly aware of that throughout my experimental career.
New harvesting methods thought about on Friday, March 15th, and continuing with this, trying to get decent chromosome. For example, on page 70, one, the arms are close, but many singles of good quality. Two, rather badly clumped, etc. Three, probably worse [00:15:00] than two. Four, some usable, but [scare?] a few small, usable arms close on five; doubtful. A few on six. Seven, clumps. But enough single arms apart. Eight, arms fine, more clumps than usual. These are different treatments in spreading.
How to look at the sample, for example on page 73, “Foggy with slides can be scanned quickly using 16X oil, and #3 diaphragm, dark field,” etc., “and then [00:16:00] direct to 100X and no diaphragm change,” etc. How to look at the result. Or pages and pages of the same type of work.
Friday, March 29th, a BUdR experiment to check which mitosis we have reached, plus or minus label, etc.
More careful check on Saturday, March 30th, [00:17:00] with a conclusion that tritiated tryptophan is too high.
Back to lambda plasmid on page 85, that’s to say, again, looking for cells in which lambda was replicated, but not lysed until we wanted to lyse them, so taking the cells and adding lysozyme buffer, and magnesium [00:18:00] so on, to isolate the DNA. Cindy did this experiment, probably Cindy Hsin. Cindy did this experiment, and obtained about 80mg of crude powder, and pronase digest gave many linear large DNA molecules, and about 1/20th of circles, but even some fields with two circles, suggest some endonuclease, tried to digest again using high EDTA and pronase at 60 degrees. “Results no better; could be worse. Try proteinase K.” [00:19:00]
Here’s the first note I have of Byron Ballou. Byron, this is Friday, May 3rd. Byron made urea 0, 2, 4, 8M urea, protease K, and pronase — that’s probably proteinase K, digests of hemoglobin under the same conditions, etc. Conclusion: not much digestion.
Page 89, lambda dv circles can [00:20:00] be added to digest these controls for endonuclease. And Byron is labeling experiments, carrying out the experiments with HeLa cells on page 91, Monday, May 27th. Using [have?] fluorescein, looking for cell survival of HeLa cells under various conditions, and using [diacyl?] fluorescein, [V37?] [retropy?] to look for living cells, and dead cells. [00:21:00]
And, talking to Harry Eagle about labeling of HeLa cells, with notes on pages attached. “Harry Eagle (by phone) recommends use all essential amino acids at greater than or equal to 0.05mM, tenths usual,” etc., etc.
And new HeLa cells from Jane Huberts, on Friday [00:22:00] May 31st. Some comments written in magic market, “MDP, Dave Poulik, 100mg/mL of beta-2 microglobulin, put through a column.” These are, it says, 50μL labeled at time, but it doesn’t say what was the label. [00:23:00] But anyway, the aim was precipitation of beta-2-M, and [congenus?], page 99, Friday June 21st, “Aim to precipitate beta-2 microglobulin, and HLA/beta-2 microglobulin from cell culture, and dissolve and separate the subunits for sequencing. Use rabbit anti-beta-2-M/goat anti-rabbit IgG, and test with I-125 label beta-2-M recovery,” so this was experiment with beta-2 microglobulin, but the reason for doing [00:24:00] these experiments is not stated.
There must have been radioactive thymidine, or no, test with iodine-125 beta-2-microglobulin, which I must have got from Dave Poulik. Anyway, these are experiments of that type, trying to isolate beta-2 microglobulin labeled in different ways. [00:25:00] And these experiments may have been related to the attempt to map the gene, coding for beta-2 microglobulin, which was a later publication of which I was part, and I’ll look that up.
So now, we’re coming to Monday, June 24th, the day after my birthday, page 105, page 104, in labeling beta-2 microglobulin. Whether this was [00:26:00] related to a later publication two years later of assigning beta-2 microglobulin to human chromosome 15 with Raju Kucherlapati, I’m not quite sure what the purpose of labeling the beta-2-microglobulin was at this point, but it may become clear. So this was I-125 labeling of beta-2 microglobulin, with two labels from Byron, that’s presumably Byron Ballou, cells in supernatant of isoleucine-labeled, [00:27:00] but beta-2 microglobulin, tritiated, and then antibody, etc., etc. And adding iodine, this is just adding simple iodide to, what at that time was a standard method of labeling with I-125 which I don’t remember anymore. [00:28:00] So, I got iodine counts of 1724*50, and tritiated counts of 6903*50. With ARGG, alanine-arginine-glycine-glycine peptide, presumably. And also another peptide, GARGG, so ARGG and GARGG. [00:29:00] And then there was Servo polypropylene tubes centrifuging at 10,000 rpm, again, precipitates. “Precipitates can be decanted, but then separate from the wall,” etc., etc., “Precipitate very gelatinous. Dave couldn’t solubilize them even in 10% [00:30:00] SDS chloral hydrate, 80% formic.”
So, the conclusion from all this is talked about on June 25th, “Nonspecific effects very large, and increase with the amount of NRS versus anti-beta-2 microglobulin.” I imagine “NRS” is normal rabbit serum, “question due to complement” etc., etc., some various schemes about what to try to do better. And, clear from typical Dave Poulik results that all the iodine-124 beta-2 microglobulin [type?] are not bind-able. [00:31:00] So, this was an attempt to get radioactive beta-2-microglobulin, I think to give to Dave Poulik for some experiment. “Clear than anti-beta-2 microglobulin is much diluted, or otherwise, no precipitate with ARGG at any of the dilutions.” I don’t know why I expected precipitation with ARGG, that’s a peptide. A comment that, “Only 20% of the I-125 is precipitable, and [00:32:00] should be more than 80% to be useful.”
A retitration of the anti-beta-2 microglobulin with ARGG. “ARGG” is evidently not what I think, because goat “ARGG,” anti-rabbit gamma globulin, that’s what it is. So “GRGG” is goat-anti-rabbit gamma globulin, and “ARGG” is anti-rabbit gamma globulin. And NRS is normal rabbit serum. So let’s go back again and pick this story up from page 105. [00:33:00] So I’m going back to page 105.
This is an experiment, beginning to take tritiated beta-2 microglobulin, and label it with iodine, I-125. And, so the experiment is starting with radioactive iodine. And I got the tritiated isoleucine-labeled beta-2 microglobulin cells in supernatant from Byron, presumably Bryon Ballou. [00:34:00] And I tried to separate the tritiated thymidine, and the iodinated material, with in the presence of increasing amounts of normal rabbit serum. Some rough curves, of doubtful (inaudible). Anyway, the precipitate was very gelatinous, and not easily dispersed. Dave couldn’t solubilize them even in 10% (inaudible), or in the dimension of chloral hydrate, 80% formic, which is Byron Ballou’s method. And so, I’m not surprised [00:35:00] that Byron was in this. He was a great fan of chloral hydrate.
The conclusion on June 25th is that nonspecific effects were very large, and increase with the amount of normal rabbit serum. Those are anti-beta-2-M, etc., etc., problems.
Continuing to try to clean this up on June 25th, with a comment clear from a typical MDP result that all the I-125 beta-2-microglobulin counts are not bind-able. So it’s not very good.
Continuing with that sort of comment on page 117, “Clear that anti-2-beta microglobulin is much diluted, or otherwise odd. No precipitate [00:36:00] with anti-rabbit gamma globulin at any of the dilutions, but progressively greater, as expected with normal rabbit serum. Must have better material available,” so on and so forth.
Re-titrating the anti-beta-2 microglobulin with anti-rabbit gamma globulin on page 119, centrifuged in [photograph?], can’t see anything. Repeated the titrations, all look about the same, on Monday July 1st. 1/5 of the anti-rabbit gamma globulin is approximately equivalent to 1/20 anti-beta-2, or normal rabbit serum, [00:37:00] images of precipitate in the tube. Quite easy to see in the images. And in the interpretation of the images on 121.
So continuing to try to label the material on the following pages. Attempt to check the H-115 failure, and with conclusion, “Discarded this tube of antiserum. Retrying isoleucine label,” Tuesday July 2nd. [00:38:00]
Wednesday, July 3rd, a repeat of the H-127 experiment, “Something wrong,” etc., “Main trouble: too few counts.” [00:39:00]
Other entertaining mark on the top of page 133, Thursday July 4th, “Ah, I see what it is.” It’s talking about the firework display on the 4th, a little diagram, a little image of a firework display. The experiment was a trial of the sucrose cushion, etc. And testing it on Friday.
Getting a little bit better, Friday July 5th, “Conclusion: the recovery is better, but the cleanliness is worse at 2X.” [00:40:00]
Continuing with Byron’s beta-2 microglobulin, labeled with isoleucine. “[Intact?] titration of the anti-rabbit gamma globulin (anti-rabbit Fc),” perhaps that means “anti-rabbit constant region Fc,” yeah. This is goat anti-rabbit Fc, unabsorbed, but decomplemented from [decon?] about July — so, and ARFc is anti-rabbit Fc fraction. [00:41:00]
Continuing in this general way with page 146, “Recheck for chloral hydrate.” Dissolving material in chloral hydrate. [00:42:00]
And then on the next page, Tuesday August 20th, page 149, the adding of the pages “chloral hydrate” again, with a note that, “Can have 10% volume/volume of chloroform in place of 10% water if desired,” etc. So this is a chloral hydrate gel with ammonium formate, and made to pH 3.5-7 with formic acid. And, then, I’m making a polyacrylamide gel, acrylamide is added, and bis-acrylamide, which can [00:43:00] be kept for several weeks at -20, and longer at -80.
And on the opposite page, hydrogen peroxide method to get polymerization in less than or equal to two minutes for a stacking gel, that’s formula for a stacking gel, etc., and tray buffer, so making a chloral hydrate gel.
And testing, on page 151, with a comment that, “This is a starch gel system, and the starch dissolved [00:44:00] the chloral hydrate; it dissolves the starch gel.”
And autoradiographs to calibrate the I-125 label. [00:45:00] And set up a complicated test of different films, with x-ray in the pack, 3,000 in the pack, and Polaroid-3000 in the pack, and [Saran?] and test paper, etc. It’s a quite complicated stack (inaudible), to extract some information out of it. And the conclusion of this rather complicated experiment was that, “Even through, I can expect to [00:46:00] detect 1-2,000 counts (inaudible) per band overnight, perhaps half this if direct contact, Polaroid-3000 is no good. Film packs, film packs, [attenuate?] [quite a region?]. Polaroid better from opposite sides from usual. So a rather complicated attempt to get labeling patterns.
The book ends with I-125 labeling with chloramine-T [00:47:00] method there, so that answered the question about how the I-125 was used, which I’d forgotten, I-125 and one with chloramine-T, and the other with lactoperixodase to label protein with 125-iodine. And that’s the end of this book.
OS:
[00:00:00] An addition to what we’ve already recorded with regard to Book h, and Byron Ballou, and the first entry being that I think I had mentioned already was Monday, May 27th, page 91. So this was Monday, May 27th, page 91. Byron labeling experiments with HeLa cells. This was beginning of attempts to label antigens on cell surfaces that could be sequenced by intrinsic radioactivity; as we shall see, a paper was published on that topic. Here we are, February ’94, and the paper I’m talking about [00:01:00] was eventually published in December ’76, was paper, “HLA Membrane Antigens: Sequencing by Intrinsic Radioactivity.” This was beginning of that work. That paper had Byron Ballou as the first author, and Byron came to work with me as a postdoc about that time, after having had a rather unhappy time in a previous postdoc. And so that was Byron labeling experiments with HeLa, that were done at that time. And, as I already have reviewed in Book h, a fairly extensive number of experiments were done to get labeling to the protein. Then I will continue [00:02:00] with a later book.