Oliver Smithies:[00:00:00] So here we are now going into the Greek alphabet. Alpha. July 1981 going up through October of the same year still in Wisconsin. So beginning with testing these various ori vector and ori tests in SVO10, etc. So I’m saying I will call ori vector alpha1oV and ori test oT3.1 picking clones and nick translating the pieces. Minis for the ori test in the following days. Autoradiograph of the minis on [00:01:00] page nine and four very strongly hybridizing minis against the probe that I had chosen. Repeating the digest on page 11. And bulk growths on page 13. Another pad may procedure protocol talked about on page 14 from Dick Flavell’s lab. Handwritten protocol from that lab. [00:02:00] Before the xeroxing. So on Tuesday, September 29th, page 19, subcloning nonrepetitive 770-base-pair fragment from 3.1-kb plasmid with A gamma delta bipolar Alu sequence in it. So looking for the bipolar Alu between A gamma and delta. And here’s the first mention that I’ve come across of Nobuyo who had joined the lab. Nobuyo Maeda, whom I later married. Found that the [00:03:00] in F 1 fragment is the best single copy piece for the bipolar Alu region. So I asked her to help me do this type of work. She came from Walter Fitch’s lab. Happy accident for me that she was due to go to NIH but President Ronald Reagan froze all appointments at that time because of a budget consideration and so her second postdoc which was to be with someone at NIH evaporated and Walter Fitch, with whom she’d been working, didn’t have any money, and so she came to work in my lab after being recommended by him. I always say the joke that she was recommended as being very polite and listening carefully to [00:04:00] what you had to say, and then do what she wanted. Well, as I’ve also said, that proved to be partly true, but no longer does she listen to me. Anyway here she is. She is beginning to work on the origin of replication. So Wednesday, September 30th just proceeding with cloning this DNA. Stockmayer by Jake on page 27 for pBR322. I think it’s the only summary of the Jake calculation that I have available. [00:05:00] I don’t think the programs exist but at least it was what concentration should be used for the target and the vector when the vector was pBR322. So here we are on Sunday, October 4th. Set up 23 tet minus and plus colony for minis. That meant they were sensitive to tet but resistant to ampicillin and there were a whole bunch of these colonies set up. But with a note that [00:06:00] the probe was from a plasmid and so every one is positive, typical mistake.
Wednesday, October 7th, page 37, S1 nuclease assay for related sequences. Aim to distinguish quickly the equivalence and assess differences between two closely related sequences. Was an idea here on how to do that. More specifically set out on the following page 39. October 8th, plasmid digest chromosome A and chromosome B talked about. [00:07:00] Let’s see what chromosome A is and what chromosome B is. It’s probable that the A plasmid is G gamma and the B plasmid is not clear. We’ll pause. [00:07:44]
OS:[00:00:00] Now book alpha. Picking up book alpha on page 41, Thursday, October 8th. There was a little confusion about what chromosome A and chromosome B are. Those are in fact the two separate isolations of the G gamma plus A gamma on a single clone and so one was called chromosome A meaning it was the first isolation and the other was chromosome B, the second isolation. So two isolations of a G gamma plus A gamma gene. And then the plasmids considered on page 41 are Bam Bam 2.6-kilobase piece available from chromosome A and the same available from chromosome B. [00:01:00] So this enables one to see whether both isolations of the same region are the same. That 2.6-kilobase Bam Bam piece which we’ll see where it comes from. This was looking at these two Bam pieces from chromosome A and chromosome B. With the result on page 43. The digestions are adequate but need a longer time. And see what the final conclusion was from that. [00:02:00] So that was an intermediate step on page 43. Let’s continue to see whether that makes sense later on. Continuing labeling experiments. [00:03:00] And not worrying anymore. And going on with new origin tests on page 51 Wednesday, October 14th. Dixie Mager and OS had an idea on how to test for origins there. Fairly complicated procedure with at least five steps. That was an idea, not an experiment.
Working on hybridization and S1 nuclease conditions. Friday, October 16th page 57. Back now to chromosome A versus chromosome B on page 59. Saturday, October 17th. [00:04:00] With some results on Sunday, October 18th. Chromosome A and chromosome B from earlier and testing with different amounts of and different lengths of time of enzyme by S1. But the conclusion is that considerable portion of the end label is being lost by S1. And so continuing to try to solve these problems. [00:05:00] Begin digesting probes on page 71, Tuesday, October 20th. New hybridization, etc. Considering using Taq enzyme and ClaI and PvuII. Different tests on page 75. Recut of the plasma 2.6 for end-labeling. That’s [00:06:00] 165.24, the 2.6-kilobase fragment from 165.24. With various conclusions about the end labels. There was nothing wrong with the previous material except all the ends are labeled. But the conclusion that the plasmid is in the opposite orientation of expected. So that see page 82. On page 82 is a complex diagram of p2.6 with [00:07:00] two possible orientations of 2.6 being listed there. This is reminiscent of a similar diagram that appears in the preparation of the plasmids for gene targeting where a rather complex map is written as two circles and trying to distinguish two orientations. So predicting what all the fragments would be in the two orientations and then trying to determine from it what the orientation is. So continuing with that experiment on page 85.[00:08:00] And hybridization tests, etc. It’s continuing to hybridize again on Monday, November 2nd, page 93. And a fairly extensive test on page 99 and 89 saying that it’s a very bad gel but clear that either S1 mix is OK, and the sites are compatible with the following order. [00:09:00] So here was the order of the sites for — more tests on the following pages. S1 tests on the 5’ end November 9th. Just technical problems. New hybridization conditions Tuesday, November 10th, page 109. Problem is the sample. [00:10:00] Permanent denaturation tests were considered, a method of permanently denaturing maybe with glyoxal. So that this is on page 117. Alkaline denaturation. [00:11:00] With sodium hydroxide. Then adding glyoxal. And with a conclusion on the following page that alkali load is sufficient. Boiling post S1 seems unwise. And formaldehyde plus alkali is partially effective presumably because of the alkali. Further tests continuing. Time series on page 123. Here’s a mention of Paula. That’s my first remembrance of — remember Paula Henthorn, a graduate student. [00:12:00] Yes, I think this is the first mention of Paula Henthorn. And she was working with thalassemia chromosomes to try to find out whether thalassemia — where the crossovers were in generating various deletion type thalassemias. And later on in looking at delta beta type of thalassemia. Very interesting work. So here is test. [00:13:00] With Paula’s material. Looking to find the end of the deletion was the aim.
Test of chromosome B at last with three exclamation marks on page 127. So this is a separate isolation of the G gamma and A gamma region. So this was a test of two different preparations. [00:14:00] A preparation from book alpha page 89 of chromosome A SST Bam fragment. And alpha 41 chromosome B SST Bam. So these were the same fragment from the two chromosomes. Trying to find out whether they differed at all. So they’re referred to in their various positions. So alpha 89 is preparation of cold SST Bam for chromosome A. And alpha 41 I presume that’s the same. [00:15:00] But we look back. And chromosome B has SST pre and SST Bam fragment alpha 41 0.1 micrograms per microliter of that. So these two preparation of the same fragment from the two chromosomes were being examined. The conclusions that the predigestion samples of chromosome A and no enzyme of chromosome B show signs of aging but there are no detectable differences by this S1 test on page 129. So that so far no difference.[00:16:00] That would be only difference really in length probably. Continuing. The same type of experiment. With comparing chromosome A and chromosome B fragments. On page 137 is a summary of the results to this point in the form of a diagram of what was being looked at. So more stringent tests are being considered [00:17:00] to look at the G gamma and A gamma genes of chromosome A and various ideas on how to test the differences.
Rather interesting title. Cold cut. Wednesday, November 25th, page 139. G gamma TaqI and A gamma TaqI and PvuII tests. New hot probes on the following page. Orientation of the new probes being looked at. And material that was made on page 143 [00:18:00] Wednesday, November 25th was with very hot material, 100,000 counts per minute per 0.08 micrograms of alpha 141 A gamma star Fnu. These were made by Sixiang. He made on page 141 5’ end labels of RI cut G gamma and A gamma plasmids. [00:19:00] Obtained G gamma 1.8 times 10 to the 7 counts per minute in G gamma. And A gamma 1.08 times 10 to the 7th, etc. And these materials were further being considered on page 143 and 145 Wednesday, November 25th. Conclusion. This material is very hot. Use fewer counts by type etc. and increase sodium hydroxide. But very hot radioactive material. Further checks on the new probes October 27th, Friday, page 147. The probes are fine. [00:20:00] Though G gamma should have been cut with PvuII. Probable trouble was too much probe DNA. Test with less probe. Cutting out bands to get specific probes on November 30th, Monday, page 151. As we come to the end of this book with some cold cuts to end. End of book alpha.