You have all heard about the DNA double helix and genes. Many of you know that mutations occur randomly, that the DNA sequence is read by successive groups of three bases (the codons), that many genes encode enzymes, and that gene expression can be regulated. These concepts were proposed on the basis of astute genetic experiments, as well as often on biochemical results. The original articles were these concepts appeared are however not frequently part of the normal curriculum of biologists, biochemists and medical students. This course proposes to read study and discuss a small selection of these classical papers, and to put these landmarks in their historical context. Most of the authors displayed interesting personal histories and many of their contributions go beyond not only the papers we will read but probably all their scientific papers. Our understanding of the scientific process, of the philosophy underlying the process of scientific discovery, and on the integration of new concepts is not only important for the history of science but also for the mental development of creative science.
Part 5
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來自 University of Geneva 的課程
Classical papers in molecular genetics
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從本節課中
Session 1
At the dawn of genetics, in the work of Mendel and Morgan, there was a complete void between the genes and the characters they determine.During the first week, we will discuss the relationship between genes and enzymes. We will start with the description of alkaptonuria by Garrod, in 1902, which he called a few years later an inborn error of metabolism. This was the first documented example of a human recessive trait, the first association of a human condition with Mendel’s principles and the first link between a gene and an enzyme. This work and that of Cuénot on mice fur color were essentially forgotten in the biology community in the following decades.After working with great difficulty on the enzymatic cascade that leads to the formation of the pigmented eye of fruit flies, Beadle and Tatum founded the field of biochemical genetics by isolating conditional mutants that affect the synthesis of vitamins and amino acids. This was first done with a mold, and then extended to bacteria. These experiments lead to the “one gene, one enzyme” hypothesis. While the hypothesis is now proven in many cases, the exceptions, including multigene enzymes, structural and enzymatic RNAs have expanded the concept rather than invalidating it.
與講師見面
Dominique BelinProfessor
Department of Pathology and Immunology University of Geneva Medical School
[MUSIC]
Beadle went to a lecture given by Tatum on bacteriology.
And there he listened to Tatum explaining how you can distinguish different bugs
on different plates, on different petri dishes with different nutrients and so
on and so forth.
And that's where the larvae as a petri dish came as an association.
And he had this insight to change and
not do what everybody had done before to start from
a genetic trait and go towards the chemical reaction.
But to start from a chemical reaction and go to the genetics to make mutants.
And this is a completed version of the ideological
background way of thinking and way of doing science.
And they started with a little mold called Neurospora.
You've all seen Neurospora, if you leave your bread staying on the table for
too long, you will find little blue, white mold growing.
That's Neurospora.
Why Neurospora?
Well, because Beadle had a friend and colleague at Caltech
who was working on Neurospera, so he knew a little bit about it, one reason.
Second reason, Neurospera had a sex life, so you could do genetics.
And third reason, bacteria which were much simpler and
easier to use, were not believed to have a sex life.
We'll, actually, at that time not even believed to have genes.
So those were three good reasons to start with Neurospera.
Of course, in the later and more successful years,
they used essentially only E.coli.
So, what did they decide to do?
They decided to do this reverse thinking,
this start from a chemical reaction and go to the mutants.
Well they don't pick any chemical reaction.
What they said is they're going to consider
that there is a series of chemical reactions that are all necessary
to make the building blocks of life.
Amino acids, vitamins, basis of the DNA and RNA.
All these little molecules that are required to make big molecules,
macromolecules.
So they know that
the chemical reactions are controlled in some manner by genes.
It's interesting to note that there's one thing in common with
is they talk about chemistry.
Biochemistry doesn't really exist yet.
It's chemistry.
They say that, Genes are part of the system, and they control or
regulate specific reactions in the system either by acting as enzymes or
by determining the specificity of enzymes.
At that time, 1941, people had no idea what genes were.
Most people believed that genes were proteins, made up of proteins.
And proteins were enzymes, so it was not unreasonable
to propose that the genes and the enzymes are the same kind of molecules.
But, they are smart enough to say that maybe the genes
do something that make the enzyme do what they do.
Determining the specificity of enzymes.
They are totally aware that there must be orders
of directness of gene control ranging from simple
one to one relations to relations of great complexity.
They are totally aware of that.
Because they know that there are mutants that affect development,
that must affect a large number of different things.
They know that the mutants that can be attributed to
a single chemical reaction are only, were only at the time.
So they know that.
Imagine that some genes are probably what we call today master genes,
regulatory genes that control many other genes.
But they must be genes that have a one to one relationship with enzymes.
And they discussed this because there is a work that has been done in plants,
mostly in plants color.
The color of the petal
that suggests that single genes control single axiomatic reaction.
Again this is a very tedious and
very difficult work that it was done by looking at the color of the petals and
isolating the pigments and showing that the pigments differ from one
another by one metal group, one hydroxyl group, one chemical group.
A simple chemical difference.
So they know that in the plants, but the plants are very hard to work with.
And again, in the plants, you're talking about the nth product, the phenotype, and
going back to the chemistry.
So what they want to do is start with the chemistry, and
say we're going to pool all the genes that code for enzymes.
And we're going to see whether we can mutate them.
And so, for this,
they will use a petri,dish.
And the petri dish and only Beadle or Efflusi could have thought about that.
Some petri,dishes have a rich medium that can help the disk to develop normally and
some cannot help the disk to develop normally.
Some are poor media.
Rich medium, poor medium.
Beadle had that in his own hands.
So, they setup to say okay, we're going to take everything that is in the rich medium
and remove everything that is in the rich medium, to make it a minimal medium.
And then we're going to talk about something which
are in English called dispensable functions.
Now, dispensable function in English is related to the word dispensary.
The place where you can buy drugs or chemicals.
In French, it's a little bit more difficult because [FOREIGN].
You use only the word [FOREIGN], which is a very old word for
dispensary, for pharmacy, which is not used anymore.
So when you say [FOREIGN], it doesn't help.
In English, dispensable functions is what you can get
from the shelf in the supermarket.
What you can get from the medium in the rich medium.
Of course, they're aware that if some goodies are required but
cannot cross the cell membrane, it's as if they're not dispensable.
Because it's as if they're not there, because the cell cannot utilize them.
But they know this notion of rich and poor medium.
So that's what they're going to to do.
And they're not going to look at the cart, the morphological cart,
as the color of the eye, the shape of the wings.
They're going to look at simple growth or no growth.
And that's going to be the Genetic Control of Biochemical Reactions
in Neurospora, by Beadle and Tatum at Stanford.
So, you have to know a little bit about the biology of
neurospora to understand the paper.
This is what they will look at.
This is what growth in the tubes.
It's called the haploid branch coenocytic.
Coenocytic means there are many nuclei per cell, okay?
What they are going to also use in the paper are this asci.
Asci are a little bag that has individual haploid cells, haploid spores.
That's what they [INAUDIBLE] to you.
So, how are they going to use it?
They're going to take a culture of normal Y type neurospora,
and they're going to take a culture of neurospora that is irradiated with x-ray.
Hermann Muller, in 1927, had made a breakthrough for
which he got the Nobel Prize by showing that x-ray can induce mutations.
And we'll talk about that later when we talk about Max Delbruck,
x-ray induced mutations.
They irradiated both together.
And they mate.
So they form zygote.
These zygotes are grown in rich medium.
Everybody that can grow will grow.
And then they do meiosis so that they have these fruiting
body from which they isolate this bag, the ascus.
This bag can be opened, and
each of these individual spores can be grown on rich medium.
Some will not grow, tough luck.
But some will grow.
Beadle and Tatum decided set for a number.
They said we're going to look at 5,000.
To see if our hypothesis is true, we're going to look at 5,000 spores.
And what we'd want is things that will grow on rich medium.
And will not grow on poor medium.
So we're to transfer these spores onto two plates,
a rich plate or rich tube, and a poor tube.
The cells that don't go on the poor tube that means that they
cannot make something that can be dispensed by the rich medium.
It's very simple, it looks trivial.
I know it looks trivial, but it was an amazingly smart thing to do.
So, rich and poor, they said we're going to do 5,000,
after 5,000, we don't see anything, we quit.
And they were lucky because spore
number 200 and I don't remember,
I don't remember which one, I think it's 227 or 207,
97, something like that, before 300, they got a mutant.
Well, they got a strain that will not grow on a minimal medium, and
will grow on the rich medium.
And in fact, they got three different strains with three different requirements,
so the observation was a general one.
Again, the generality of the observation.
They had not just shown one vermillion cinnabar,
which is a beautiful piece of work, but is limited.
Now they're going to do something that anybody can do
with any microorganism, isolate conditional lethal.
Lethal on minimum medium growing on rich medium, it's called conditional lethal.
We'll come back to conditional lethal later.
So they isolate three conditional lethal mutants.
One requires vitamin B6,
one requires thiamine or benzoic acid,
and one requires vitamin B1.
Free vitamins, by chance.
We're not particularly interested in vitamin metabolism.
It's just by chance these vitamins are present in the rich medium,
absent in the minimum medium.
Okay, so they only describe in the paper the B6.
But what is true for the B6 must be true for the others.
Hence, generality.
Okay, so.
So what do they do with the B6 mutant?
Well, they set up an assay for
growth, That is, a quantitative assay.
And they describe in the paper, and
it's sort of amusing, they describe that they're using long glass tubes
that can be as short as 90 centimeters and as long as one meter and a half.
As thick as 1.5 centimeter or 0.6 centimeters,
with the ends blocked or not blocked, so there's enough air.
They describe the system in such a way that you can build
the experimental tool to do the study.
And that's not always the case in the papers we will read.
But at this paper it is described within extremely great detail.
And so they measure the rate of growth in centimeters per day.
The unit is centimeters per day, or for growth.
So they're going to have times, days or hours and centimeters on the y axis, okay.
Now this is a slightly unusual graph because what they have is 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13 graphs on the same plot.
It's a bit unusual, okay.
So what did everybody starts at zero in centimeters and
then everybody spread on the Y-axis.
So you can see all the curves.
I've never seen this kind of drawing being used afterwards.
But it's a nice way of drawing things.
So the first two are black ones, are the normal strains.
The normal strain grows at a certain speed, whether you have vitamin B6 or
you don't have vitamin B6.
Now this is luck because sometimes when you have too much
of a goody, you can grow less well.
If you have a too rich meal, you have a hard time walking up the stairs.
That happens.
In this case, you can add one microgram of vitamin B6,
and you grow as well the as without.
What about the mutant?
If you give the mutant to one micrograms per mill, per 25 millilitres here,
it grows as well as the y type.
If you don't give any vitamin B6, it basically doesn't grow.
Now, remember that it is not necessary
that the mutant supplemented with the vitamin grows at the same speed.
That is not always necessarily the case.
Because if you take a mutant and you have a chain of reaction that makes a number of
intermediates, and what you mutate is this enzyme.
If you mutate this enzyme, you don't make this reaction, right?
So, whatever was here, the intermediate at this step,
will accumulate in the cell, and may be toxic.
Even if you add the vitamin B6 from outside
you still may accumulate these things inside.
And so, the fact that it grows normally was not is nice, but
is not necessary for the argument.
It's just nice.
So, they have this mutants and then they will do another thing with this and
they will analyze the gross in gross per day,
centimeters per day versus concentration of vitamin B6 and you get this curve.
Whether you have point two five five point five one, it's about the same growth.
You saturated the system.
If you don't have vitamin B6, you don't saturate the system, you don't grow.
Now, you don't grow why?
Because you need this vitamin.
But you still grow a little bit, even without the vitamin,
you still have some little growth.
What is that?
Well, there are many explanations, many possible explanations,
none of them have been tested, this work.
One is that the mutant is not a complete now, it still works 1% of the time,
that happens.
Second possibility, the goodies you put in your petri dish are not clean.
I mean they're clean, but not super clean.
And so you have traces of contaminants that can be used by the vitamin B6.
Those are the main explanations.
So this kind of an acid is still used today to titrate vitamins.
When you go to the favorite store and buy A to Z, vitamin A to Zinc,
you buy vitamins that have been tested and quantified by assays such as this,
because if you use this concentration of vitamin B6,
you have about half the maximal growth, so this is a typical.
So they have, the biochemistry is vitamin B6.
It's not yet the one gene-one enzyme.
It will lead to the one gene-one enzyme because it is the one gene,
one pathway, one biochemical pathway, the one that leads to vitamin B6.
Or the one that leads to para-aminobenzoate, and for vitamin B1,
which can be cleaved in two, in two parts.
They have shown that it is one of the part and not the other part.
So it is close, but not yet to the one gene one enzyme.
But this paper is taken by other scientists
as the founding paper of the series and
then there have been hundreds.
Now let's go to the genetic because so
far we've done the biochemistry.
And that's about all we can do at this stage.
They want to know whether this strain which has been isolated
after x-ray mutagenesis,
is a genetic defect that can be isolated in a cross.
Because that's what genes are defined.
The way genes are defined, so they will make cross, so
they will cross the mutant with the Y-tap.
So they cross, they make the deployed,
and then they make the ascii and then they take the individual's force.
One to eight and they tried to let them grow.
And 16 of the ascii do not yield any viable spore.
It's only when they reach 17 that they get some spores that grow.
And they stop at asterisk number 24, which behaves as if
the mutation is on a single gene or on a single chromosome.
It's very poor genetics, if you think of it because
they stopped with a single ascii that behaves properly.
None of the other behaved properly which is very bizarre.
And so they are, so they have.
But they're fair.
They report their results.
And they don't understand at this time why this is so.
Why do they have so many spores that are dead?
What they didn't know at the time is that x-ray
induced chromosome break and reunion chromosome translocation.
And that is something very tricky because when you do
a chromosome translocation,
it's as if you take one part of a book, and put it in another book.
And you exchange.
Okay, that you can imagine.
You take out a part of the book, put in another book.
You take some part of the other book and put it in the other one.
Okay, you still have all the information but it's not in the right order,
in the right books, but the spore has all the information.
So if this is a normal spore with two chromosomes and
this is a spore with a translocation, with chromosome t translocated one and
translocated two which are different from N1 and N2.
When you do meiosis, you will put together
pairs of chromatids, single units of chromosomes.
Now this is a bit of a mess because
the translocated portion will go with it's cognate,
the normal goes to where the normal is etc etc and you get this complicated figure.
You can draw this complicated figure yourself with four strands of T1N1 T2N2.
This can break at meiosis and at meiosis,
you will have different chromosomes.
If the spores are like this with the adjacent transfer locations,
each of these spores will lack something.
If you have chromosome T1, you have all of this.
If you have chromosome N2, you are missing part of chromosome N1, you're dead.
All of these guys are dead.
If you segregate properly, you can get the normal chromosome T1 T2,
N1 N2, and these are all okay.
This is askis number 24.
Everybody grows four mutants, four white that.
All the others are like this or sometime like this when you get four spores
that don't germinate and four that grow.
Now this is with one translocation.
Now if you are prepared to spend some time and have many colour pens,
you can make two translocation on two different chromosomes, so
that would be four chromosomes and you can draw all the possibilities and
see how many are viable, how many are not viable, right?
It is estimated that in this case, they were between two and
three translocation in their mutants.
So it's not unreasonable that they got many, many,
many active spores that died after meiosis.
