Last time we have already discussed in detail about, maybe the toxicology and about chemical approach to identify the key molecular machinery, namely the snail complex, okay? The work originally people identified synaptic writing so the neurotoxin assay, understanding a substrate. And then Gene Rossman, his group, using the biochemical approach purified the NSF, and ATPase that is critical to understand to likely transmit and reduce and identify its membrane receptors, the so-called snap receptor, snares. And it turns out they are the same, okay. And we are going to discuss briefly yet another approach which is using the genetic approach. And one of the classical papers listed here conducted by Randy Schekman. Still not in Berkeley. Okay. So in early 1980s this is a cell paper. Identification of 23 complementation groups required for post-translational events in the yeast secretory pathways. So Randy is a well-established biochemist, even though his classical work that we will discuss today is based on genetics. Okay. So, he and Jim Rossman, actually, also share some background. They were out, from the Stanford biochemistry department. So Jim Rossman has served, in the past as a chairman, for the biochemistry department at Stanford. Where Randy Shekman was a grad student, in that department. Working with, Arthur Cumbrick. Another great American biochemist, who also got his Nobel prize by identification of DNA polymers. Okay, so, both Randy and Gene Rossman that we discussed last time, they have the history, all been associated with Stanford biochemistry department. If I'm aware they use this biochemical approaches to understand. Where this paper that Randy and his technicians and students, they designed a very screening acid. Okay? So again, as a biochemist, I was joking saying they have this is three essential tools. Three essential kits in their hands to ascertain different biological processes. Okay, one is running a jowl, right, I was joking, the other one will be centrification, right? If you still remember. The third one will be, run some kind of columns, okay? Well, in this case, what it did actually, is using the yeast genetics. So they demonstrate that using a special centrifugation process, they can separate the mutant yeast versus the wild type yeast. So this is the different collection of the centrifugation that separate the special yeast cells by their density. Okay and here demonstrating a mutant yeast they previously identify that has abnormal vacuum. Okay, it turns out the yeast has vacuum and if you have this abnormal vacuum. The yeast density is different that water, okay? So in their acid as you can see that most of this fracking here listed are all the well type, has a normal density. Well there is this mutant, this has very different ones and then can be clearly cut, separated. Okay? So in their case so they were thinking well if they can conduct mutagenesis in yeast, and then using this [INAUDIBLE] protocol, just by centrification in a separate different strain of yeast despite that physical density. Then you can collect a lot of mutants. And better than that, what it did, using the powerful yeast genetics is they conduct a temporary geo-sensitive screening. Meaning that only the yeast that in our now optimal temperature if they have their difference in density, they will collapse. In the optimal temperature, the protein even though it is mutated can still be functional, and therefore can support the yeast to grow. So in this way they can generate a screen that can identify those essential gene that might be important for survival of yeast by simply screen in a different temperature. While maintaining them in a optimal temperature while the protein at least is partially functional. So, this screen, they identified many, many mutants. So this is demonstrating that using their enrichment method. And TS means the temperature sensitive. So they can screen many different colonies in which as enrichment method as you can see, that they can get. For example, close to 200 mutants were in this without enrichment, you only got 2, okay? So this weight, they can enrich it. So here's a problem, after your screening identify twin, more than a 150 mutants and they are all temperature sensitive mutants. The problem is how do you know whether they are hitting the same gene. Okay. In their title as in here they demonstrate that using this EMS, which will somehow modify DNA nucleic acid and introduce single point notation. The modified DNA actually goes through the DNA replication. You will, can mutate it. So they identify the so-called second mutant, second with secreting, okay. Circulatory secreting mutant, second mutant. As you can see this say, the count is at 23 different mutants out of this more than 150 colonies. So how do you know? They are belonging to those 23 groups rather than more 150 different groups. How did the authors know? And if you were lead how do you know?
