Recorded: 01 May 2000
Jeff: When we cloned the mating type locus, we didn’t clone the mating type locus as a matter of fact. What we cloned was one of the silent copies. I designed a selection, a screen, for the mating type locus which involved complimenting a mutation at the MAT locus that would change the mating characteristics of a strand. So we went through a large library of clones in a vector that we made specifically for this purpose, one of the first cloning vectors for cerevisiae, called CV13, after Cadillac Vector, which was renamed by David Botstein into F13 (Yep13??).
When we cloned that, there were several problems with the clone. The first was, it wasn’t MAT. It was one of the silent copies of MAT. Secondly, it was a fairly big clone so we had to trim it down. Of course the nature of the demonstration that it was the mating type locus and not some clone which suppressed it for non-specific reasons, the phenotype that we were exploring was to do a southern blot. It was RFLP mapping—restriction fragment length polymorphism mapping—and we were one of the first people to do it actually using that clone. The previous- first example, I think, was the canr locus—the arginine permease locus, where we demonstrated that we had the arginine permease by that technique.
Anyway back to MAT, we had this clone that we used as a probe, then to digests of MATa cells and MATα cells. Of course, the cells weren’t isogenic a and α, they were just an a cell from the collection and an α cell from the collection. And when we used the probe, because it came from HMLα, not from MAT, it lit up all these other bands, and there were restriction polymorphisms for those other bands, and one in particular, which was very homologous to the probe—had extensive homology to the probe—had this big band shift. So we were reading across the gel. There were about twelve lanes or sixteen lanes of tetrads that had this mating type locus segregating two a’s and two α’s.
So reading across the band, there was this big band shift across the band like this. And so we’d read: a, α, a, α. And then it would be α, a. It was a huge disappointment, this band that was shifting, back and forth on the gel with these huge brilliant shifts reading across the gel did not match mating type, did not correspond to mating type.
We were looking at this thing, but there were three other bands on the gel. Several other bands on the gel, most of which were perfectly flat, the same in every band—gel. That didn’t matter much; there was no information content from that. But there was one band that had this little tiny shift. So after we read off the big shift, and we were like, “Oh man! We didn’t get it. What’s this all about?” We had this one little band, and I remember being in the dark room, reading off. One person had the gel and one person had the genetics in the dark room. We still were in the dark room because the gel was still wet! We’re holding it up to the light like this, reading off the gel. After having eliminated the big band, we had this little shift. So we’re reading it across like this α-a, α-a, a-a, α-α: Bands matched! I remember being in the dark room with this gel and this little tiny shift which turned out to be the right one.
So we had the agony of defeat and the ecstasy of victory—in that order—finding the perfect correlation across these four tetrads of the mating type locus clone. I remember, the fact that we were doing it in the dark room and holding the thing up to the light was really a memorable experience. As I said before, we knew we had the genetics, but now we could beat people over the head: okay, here’s the difference! And we knew we would get the rest from there. That was a pretty special day.
Amar Klar and Jeff Strathern worked together in the Cold Spring Harbor Yeast group from 1977 till 1984 where they made outstanding discoveries about the mechanism of mating type switching in yeast.
Amar Klar, is a leading yeast geneticist, concerned with the molecular biology of gene silencing and mating-type switching. Klar came from India to the University of Wisconsin in 1975 to receive his Ph.D. in bacteriology. From 1977 to 1984, he worked with Jeff Strathern and Jim Hicks in the Cold Spring Harbor Yeast Group studying the mechanism of mating type switching. Klar served as Director of the Delbruck laboratory from 1985 to 1988.
He left Cold Spring Harbor to join the ABL-Basic Research Program as Head of the Developmental Genetics Section. In 1999, Klar joined the National Cancer Institute Center for Cancer Research and is now a Principal Investigator in the Gene Regulation and Chromosome Biology Laboratory at NCI-CCR.
Jeffrey Strathern, a leading yeast geneticist, obtained his Ph.D. from the Molecular Biology Institute at the University of Oregon in 1977 and then moved to Cold Spring Harbor Laboratory, where he became a Senior Staff Member with the yeast genetics laboratory.
In 1984, he joined the ABL-Basic Research Program at the NCI-FCRDC. His research remains centered on aspects of gene regulation and genetic recombination as revealed by studies in yeast. In 1999, Strathern joined the Division of Basic Sciences, NCI. Strathern worked together with Amar Klar and Jim Hicks in the Cold Spring Harbor Yeast group from 1977 to 1984 where they made outstanding discoveries about the mechanism of mating type switching in yeast.