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From the course by University of Geneva

Classical papers in molecular genetics

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University of Geneva

Classical papers in molecular genetics

88 ratings

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.

From the lesson

Session 7

The existence of nonsense or stop codons was suggested by the mutant phenotype of certain combinations of (+-) double mutants (see previous session). Benzer had observed that certain rII mutants can behave as wild type in some restrictive strains that for example do not allow the growth of rII deletion and frameshift mutants; he called these mutants ambivalent rII mutants and the strains suppressor strains. In the first paper, he used again the deletion that fuses rIIA and rIIB while preserving rIIB function. Base analog revertible mutations in the N-terminal region of rIIA were combined with the deletion. Some of the mutations, called missense mutations, do not affect the rIIB function of the fused gene. Others, in striking contrast, abolish its rIIB function in restrictive strains but not in the suppressor strains. One of the major conclusions of this work is that the genetic code is under the genetic control of the organism itself. This control is achieved by the genes that encode the tRNAs and the amino acid synthetases of the organism. Brenner discuss the results observed in several bacterial and viral systems that describe nonsense mutations and their bacterial suppressors. They propose a unifying nomenclature for amber and ochre mutations. They show that amber suppressors only suppress amber mutations whereas ochre suppressors, which are usually weak, suppress both amber and ochre mutations. Variable results obtained with different suppressors are explained by the nature of the amino acid inserted by each suppressor as well as by context effects. The major mutagen used in this work is hydroxylamine, which can modify C residues so that they are recognized as T during transcription and replication. Since the rII genes have to be expressed before replication, a modified C will only exert its effect if it is present on the DNA strand that is transcribed. Using hydroxylamine, they show that neither amber nor ochre mutants can be induced to revert with hydroxylamine either immediately or after on growth cycle on a permissive strain. Since ochre can be converted into amber by treatment with 2-aminopurine, a base analog mutagen, the two codons must have two common bases (UAx) and the third differs by a transition (UAG versus UAA). In a brilliant hunt for forward amber and ochre mutations induced by hydroxylamine, they show that the two codons must have both a U and an A. This genetic data is combined with biochemical study of proteins produced either by suppression of amber mutants or by mutagen induced reversion of these mutants. The data fits perfectly the biochemical deciphering of the code that was performed by Nirenberg et al.

Meet the Instructors

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

[MUSIC]

So on this little diagram, you have a map of

some of the mutation used in this study.

You have the complete deletion of the R2Os.

You have a complete deletion of R2B, R2A intact.

So 638 is A+, B-.

The two deletions here are A-, B+.

And these are a series of point mutants that

are substitution of one base for another base.

And all these mutants are revertable

by 2-Aminopurine.

That is, either you can make either an AT to GC change

on their DNA or a GC280 change on the DNA.

Both are induced by terminal period and

you'll revert these mutants, so they are not insertion or deletion.

They're not like the FC mutants.

[COUGH] Here, he's basically shows again,

the experiments that were published in the previous paper,

where he identified the ambivalent mutant.

And here, he called them again the ambivalent mutants.

So the three deletions are analyzed, and

they are dead, no activity in both strains.

The ambivalent makes lysate on the SU amber host,

the suppressor of the amber code on.

And this has no suppressor, and so, in this case, there is no activity.

This is what ambivalent means.

R plus on one strain, r minus on another strain.

And there are lots of other mutants in the R2A which are not ambivalent and

the non ambivalent are these.

They have the same activity in the two strings.

There is a slight little problem in the logic of the paper at that point.

Benzer considered that if a stop codon, an amber codon, is UAG,

this stop codon can be induced to revert

by changing this A to another purine G, UGG.

It can also be used to revert by changing this U in the message

by a C and so you have CAG, two codons that code for amino acids.

Now, the argument of Benzer is that if two amino purine

can induce either one or both of these transitions and make an active protein,

the original mutant should have been either this,

the original Y codon should have been either this or this.

And this is clearly wrong because you can have, at some position,

a UAU codon which by spontaneous mutation became UAG.

You've changed the G to a U.

This is called a transversion because you replace a pyrimidine by a purine.

These two are called transitions.

So provided that the amino acid that is

coded by these triplets, is acceptable for the protein.

This could very well happen.

So you can start somewhere on the protein, and you have a tyrosine.

This is a non sens, or stop, and this is glutamine, and this is tryptophan.

So if somewhere on the protein,

at that particular place what counts is to have one residue.

But it doesn't matter which residue.

You could have the wild-type like this, the mutant, and the revertant.

So it's not necessarily, the origin of the mutation is not necessarily a transition.

So what did Benzer found?

And so, what he did, basically doing the same thing that we've seen

in the previous week, is done a complementation test.

He looked at the activity of [COUGH] the double

mutants on the R2B function of 1589.

And with one set, the ambivalent,

he finds that there is no activity in the E.coli strain that is SU-0.

And he finds activity in the SU-plus amber.

Now, what is very important in this, and this we'll

see the same with the Brenner paper, is to use a large collection of mutant.

And see whether what you observe is generally observable.

If there are some freaks, you ignore them.

Now, what's fascinating is that Benzer encountered freaks

at every single step of his work, things that did not completely fit.

In the case of 1589, did not behave the way it was supposed to behave.

And he immediately realized the advantage of this mutation for a lot of studies

In this case, he has mutants that are non sens in one strain and

sens in the other strain, but very poor.

Look at this guy.

Here, you have one phage per 1.5 phage per bacterium.

It's very low.

But, it's one of the mutant out of all the other one of the subset.

And he decided that this does not invalidate his model

because most of the other one behaved properly.

And basically, today's interpretation of this result is that when you're going to

read a stop codon, and making sense, the efficiency at which you read

depends on the surrounding sequence, what is called the context.

And the context is still not understood at the molecular level.

And it's a very difficult question to study.

But the use of a collection of mutant is very important.

All the non-ambivalent give R2B function.

All of the ambivalent don't give R2B function is SU-0 strain and

most of them do work well in a SU-plus strain.

And that, hence, the title of the paper.

A change from non sens to sens.

Because the paper [BLANK AUDIO] actually

identify a mutants which is non sens.

HB118, HB122, C204, all of these are non sens.

But you can make them sens.

Not sens in term of the function but

sens in the term of continuing translation beyond.

So this is the last paper of Benzer and now we're going

to shift to the other paper of today which is the paper by Brenner.

Now, the paper about Brenner is a little bit late later, three years later.

And it's probably the most virtuoso piece of genetic and

biochemical analysis of a biological question.

This is exemplary of what was already known at the time as molecular biology.

Combination of genetic approach with biochemical approach.

So in this paper they're going to do something

which sounds a bit strange by today's standard.

They're going to establish the sequence of the codon

by pure genetic and classical biochemical means.

Which is, now, you may think it's absurd, I mean the sequence through nucleotide and

today we sequence billions of nucleotides everyday.

But there was no machine, and there was no sequencing.

It's all deduced genetically.

So Brenner and his colleagues start by describing the principle of the code.

Non overlapping triplets from a start point.

And then, they say triplets which do not correspond to amino acids

have been loosely referred to as non sen.

Without a very good molecular interpretation of what non sens is.

And so, the evidence that they are non sens comes from in fact an argument

which they reverse, they reverse the historical order by topological order.

So they say the evidence that there are non sens

is that these mutants are suppressible.

In fact, if they had not been suppressible, they would not have been C.

They would have been mutants.

Because they are mutant on one strain and

not mutant on the other strain, that's a suppression.

And the suppression, they realized immediately,

implies an ambiguity in the genetic code in the sense that a codon maybe

have one meaning in one strain and another meaning in the other.

Benzer paper terminates by discussing

the implications of his finding for the genetic code.

And in particular, this notion that the genetic code is not something

directly governed by the laws of physics and chemistry.

Because if it was directly linked to the laws of chemistry and

physics you could not change it.

Would be very hard to change it and you can change the genetic code.

And so, the genetic code is really carried from one generation

to the next generation by a group of genes that encode the tRNAs.

The adapters of Crick.

And the that link to the amino acid to the tRNA.

That is the warden of the code.

That is who decides what the code is.

This set of genes.

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