Part 5

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来自 University of Geneva 的课程

Classical papers in molecular genetics

97 个评分

University of Geneva

Classical papers in molecular genetics

97 个评分

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.

从本节课中

Session 5

Before Benzer’s experiments, most genetic mapping required a screening of all the progeny of a cross to calculate the recombination frequencies. The power of the rII system of phage T4 resides in the facts that many independent mutants can be identified by scoring plaque morphology on the permissive host strain, and that only wild type recombinants will grow on the restrictive host strain. This, together with the very high recombination of T4 DNA, allows the detection of recombination between mutations affecting adjacent nucleotides. The cis-trans complementation test showed that the rII locus consists of two genes. Using more than 100 deletion mutants, which do not revert to wild type, Benzer first demonstrated that the topology of DNA is linear. Using these deletions, he was able to carve the rIIA and rIIB genes in 47 segments. A very simple and rapid recombination test allowed him to map thousands of point mutants, both independent and mutagen-induced, in individual segments. This map was perfectly congruent with maps laboriously constructed by classical recombination tests. The topography of the map was strikingly non-random, with sites that are 100 times more likely to mutate than others and are called hot spots. The spectrum of sites detected after mutagenesis was strikingly different. It is now known that most of the spontaneous rII mutants are frame-shift, i.e. addition or deletion of one or a few base pairs that disrupt the translation of the mRNA into protein. In contrast, most mutagens used in this work induce base substitutions that do not usually arrest translation. Now that the sequence of the rII locus is known, the saturation of the map is such that there are about one mutation every 8 nucleotides.

与讲师见面

Dominique Belin
Professor
Department of Pathology and Immunology University of Geneva Medical School

[MUSIC]

The paper that is the most known of Benzer's rII adventure,

is the topography paper, published in 1961.

Actually at the end of the paper, in the notes, he defines the terms.

Topology and topography are used here in the following senses,

from the Webster 1959 Dictionary.

Topology, the doctrine of those properties of a figure,

unaffected by any deformation, without tearing and joining.

And topography, the art of practice of graphic and

exact delineation of minute detail, usually on maps or

charts, of the physical features of any place or region.

The topography paper, In contrast to the topology paper,

the topography paper will use point mutants, and only point mutants,

as the object of study, of course, using the deletions to map the point mutant.

So this paper talks about the local differences in the properties of its part.

Are the subelements equally mutable?

Do mutation occur at random throughout the structure or not?

Now, if you think about the double helix and the model that Watson and

Crick proposed, there was no interesting features of topography,

since all base pairs were equivalent.

So, in this case, he will use a series of point mutants, and

the series of point mutants is actually quite amazing.

Because that series is made of 1,600 individual mutants.

It's one of the largest ever made.

On this slide, you can see how he was going to do the mapping.

On the top line, you have a phage map, rather crude, with different markers.

All these markers on the top, except for rII, can only be used in screening tests.

ie, you can detect whether a particle is rI mutant or

rI plus on the basis of morphology, where there is no selection.

And the same holds for all the other markers.

Only the rII can be subject to mapping,

precise mapping, because of the selection pressure and

the selection strength of the analysis on K lambda.

So if you want to map mutants, You have to do a lot of process.

Basically if you want to want map n mutants, you have to do n

square plus n over two crosses.

Over two, because crossing A with B is equivalent to crossing B with A.

So only half of the crosses are required, and

plus n because you need all the diagonal crosses, ie, the mutant with itself.

That will give you the background.

That's a large number.

If you want to use 1,000 mutants, you need, for 1,000 mutants,

you need 500,000 crosses, which is really a lot.

And Benzer was as smart as he was lazy.

He didn't want to do useless effort.

In the previous experiment he had used zero and one, plus, minus.

Yes, no.

Qualitative test.

So he thought of using this qualitative test to do mapping, en masse.

For this, he used a number of deletions that would order the mutants.

The seven deletions shown here, seven is the magic number.

There was the seventh phage of Delbruck, the seventh deletion of Benzer,

the seven dwarves and Snow White, etc, etc.

They could have used eight.

They could have used six, but they use seven.

Six of these deletion cover rIIa and rIIb.

Only the last 638 covers only b.

They all end somewhere on

the right Because in

this region of the phage they are non-essential genes that can be deleted.

None of them extend to the left,

because here is a essential gene, which is gene 60.

So you cannot touch gene 60.

Now, you agree that a mutation will be mapped easily in these segments

by crossing with the deletion.

Imagine this case, you have two deletions, 1272 and 1241, and a point mutant, x.

Point mutant x cannot recombine with 1272.

To go back to the Dante analogy,

if the book that was removed is book 34, and

this mistake is in book 34, you cannot recover book 34.

However, if this deletion removes book 35, and

this point mutant is in book 34, you can buy two copies.

One bad copy lacking book 35, one copy with a bad book 34,

and you can cross over, recombine, and make a full version.

Of course when you do this you also make a double mutant version.

Only the, Y-type recombinant will grow in K lambda, in most cases.

So this Y-type recombinant is shown here with this dotted line.

It takes a piece of DNA from here, somewhere here we change to the other DNA,

and then we continue.

This is crossing over recombination in a very simplistic way.

But that's enough for this purpose.

So if the mutant doesn't recombine, you put a 0.

If your mutant recombine, you put a 1.

Very simple.

Now all of these are drawings.

So I have to show you one actual experiment.

One series of plates.

Which look not very nice, but you would immediately understand.

If you have the bacteria growing happily,

as a lone, it looks white with this microscopic system.

It's called dark field microscopy.

If you have a virus that will make a hole in the lone,

a clear zone in the lone of growing bacteria,

this will allow light to go through.

And in this system it looks black.

It's a dark field, that's what it mean, a dark field.

So every time it's dark it means you have.

So if you take, say, mutant 1011,

and 1011 you cross it with 638, you get

because 1011 can recombine with 638.

1011 by itself, what is called here the blank, this is the self.

This will measure revertants.

This will measure the number of the background, the noise,

if you want, of revertants.

Things that happen through replication and errors and replication.

Not through recombination.

You can see, for instance, that some phage make more revertant that others.

This one makes lots of revertants.

This one make very few revertants.

This one, we can see one revertant at the tip of the arrow, one dot.

The deletions make no revertants.

So you ask, is this recombinant or non-recombinant?

This is non-recombinant, 0.

Is this recombinant or non-recombinant?

It's clear, 1.

O, 1, 1, 1, 1, 1, 1.

What's the case with the second line, that 56?

0, 0, 1, 1, 1, 1, 1.

So, the difference between these two mutants is being

capable of recombining with 1241, or not capable.

That's the difference, and so on and so forth.

And you can see I've shown you here, for instance,

this is almost certainly a revertant, and not a recombinant.

The recombinants give you a large number of plaques.

The revertant give you few plaques.

So once you've done this, you've sorted all your

mutants in seven groups, from left to right.

But he had isolated 145 deletions.

So he used a lot more of the mutants,

a lot more of the deletion, to do a fine mapping.

And I don't give you all the details of the fine mapping,

I'll just give you one example.

Here you see 1272, 1241, that we've seen the previous slide.

A mutant that recombines with 1241 but not with 1272

is defined as belonging to this A1 segment.

This is an A1 mutant.

It recombines with everybody but 1272.

Now, within A1, I can define, with two deletion, three regions.

These two deletions are 1364 and EM66.

I show you both drawings, they are the same, but some people like to see this

kind of a drawing, and some people like to see this kind of a drawing.

So you see that if the mutant recombine with both, it will be an A1a.

If it doesn't recombine with either, it will be an A1b2.

It doesn't recombine with 1364 and EM66, so it's here.

And if it recombines with EM66, but

not with 1364, you would put it in A1b1.

So with this system, with a few deletions, Benzer was able to map

all his mutants in 47 bits and pieces.

A1a, A1b1, A1b2, A2a, etc.

All the mutants in 47 segments

I show you another example from another paper,

from a paper published in Scientific American a few years later.

You have again the seven deletions, the magnificent seven,

and in this case the segment that's subdivided Is called segment A5c2a2.

In this segment, Benzer identified one, two, three, four, five,

six, seven mutants.

Up until here, this is data.

Below is his attempt to correlate

the data with a DNA double helix.

He estimated that this region is about 35 base pairs, which is approximately right.

And so in this region, he had seven mutants, so

he had one mutant every five base pairs.

Certainly not one mutant every base pair.

And you know that their letters are more important than others.

Their letter, if you remove an S from vos enfants,

or your kids, you remove the S, well,

in English, there is an ambiguity because it could be your kid singular, or

your kids, missing the S, plural.

In French, there is no ambiguity because we can say vos enfants,

and vos is necessarily plural.

If you forget the S at the end of vos enfants, everybody understands.

Of course you get a bad grade in school, but everybody understands.

So this is the kind of detail that he was able to get

in terms of what he called the fine structure of a gene.

The next question Benzer asked was, how does this way of doing,

this completely novel way of mapping, correlate with

what geneticists have been doing for the last 50 years?

Geneticists do crosses and measure recombinants.

So he had to convince, not really himself, but

he had to convince the community that his way of mapping was acceptable.

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