[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.
