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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.
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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
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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?
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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
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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.
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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
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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.
Yan ZhangProfessor
Yulong LiResearch Fellow
