Saturation Mutagenesis
Objectives
1. To understand that alternative strategies of building a catalogue of mutants versus selecting for a rare
phenotype.
2. To understand the demands of saturation mutagenesis on the vector system and the requirement for a
genetic screen.
An example: Lim and Sauer, Nature 339: 31 (1989)
Overview
Saturation mutagenesis is an in vitro mutagenesis strategy wherein one tries to generate all (or
most) possible mutations within a narrow region of a gene. The two most popular ways to make the
mutations are cassette mutagenesis, or oligo-directed mutagenesis. Both depend on synthesizing
oligonucleotides covering the regions to be mutagenized such that the oligos contain a random assortment
of incorrect bases. A third way is to conduct PCR under conditions that favor a high degree of
misincorporation.
In cassette mutagenesis, the region to be mutagenized is between two unique restriction sites such
that it can be cut out and replaced with a double stranded mutagenic oligo. When the mutagenic oligos
are synthesized, the substrate for each position to be mutagenized is drawn from a reagent bottle with a
fixed amount of the incorrect bases mixed in with the correct bases. The amount of misincorporation is
adjusted to yield some preconceived average total number of changes per oligo. This is frequently
adjusted to be an average of one change per cassette, but some experiments have been done to try to
sample all double mutations, or an assortment of totally random sequences at the mutagenized positions.
The mutagenized residues do not need to be adjacent.
The mixture is cloned, and individual clones are sequenced and subjected to characterization.
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Cataloging versus Screening
If the sought-after phenotype is expected to be found in a major proportion of the mutants, then
one often sequences the candidates at random, and then tests them for the phenotype one at a time. The
negatives are compiled as well as the positives, thus one can potentially tell that all of the possible
mutations have actually been examined.
If the sought-after phenotype is expected to be rare among a very large number of possible
mutations, then one would conduct the phenotypic examination first as a genetic screen, and then just
sequence the clones that pass the screen.
Demands on the vector host system
One wants to do the mutagenesis, screening, and sequencing in the same vector. Therefore, the
vector has to be well designed from the beginning of the experiment. If the target sequence is a ciscontrol sequence, then it should be fused to an easily screened reporter gene. If the target is an element of
a protein sequence, then the activity of the protein should be monitored as directly as possible in a genetic
screen.
Hydrophobic Core of the Lambda Repressor
The following is an example of a superbly designed experimental system. The lambda repressor
(CI) was subjected to saturation mutagenesis of the set of amino acids composing the hydrophobic core.
These were 7 noncontiguous residues that were mutated within the context of a 102 residue cassette as in
the figure above. The degree of misincorporation was such that variants at between 1 and 4 of the 7
amino acids were recovered.
Selection for function of the repressor was done by infecting the cells with a strain of lambdaphage
carrying a mutant repressor gene. Consequently, clones expressing nonfunctional repressor were killed by
the lambda phage, whereas those expressing functional repressor were able to repress the lytic functions of
the phage and survive.
The mutagenized repressors were driven by an inducible tac promoter. Some were able to repress
the incoming lambda phage when expressed at the basal level, and others could only successfully repress
if the tac promoter was induced. Therefore, the mutants were divided into three classes: totally functional
(similar to the wild type); partially functionally (requires over expression); and nonfunctional. 30-100
mutants of each class were sequenced.
The scientific result of this experiment was that you can make a lot of changes in the hydrophobic
core as long as the residues are still hydrophobic and the total volume of the core is not much changed.
The technical aspect of the experiment that I would like to emphasize is the placement of the
selection before the sequencing. Only about 1% of the mutants survived the screen. If they had
sequenced at random, and then tested the phenotype later, they would have obtained most of the data from
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the nonfunctional set. The application of the selection first allowed them to pick similar numbers of
mutants to sequence from each set.
One demand imposed on the vector in this experiment is that you make the mutations directly in
the expression vector. That means that they had to do some engineering in advance to create the cassette
in an appropriate expression vector. Another demand is that expression from the vector is at a suitable
level to conduct a genetic test. In this experiment, they used the fact the over expression can alter the
phenotype to their advantage. Ignoring this fact could lead to some seriously irreproducible results (such
as reporting mutants as wild type and then finding out that they fail to function in other assays).
Now imagine doing such an experiment only conducting the screen in a eucaryotic cell. The
demands on the vector will define what I mean by a true shuttle vector. If you tried to do this with an
acute transfection in COS cells, you could never separate the positives from the negatives, because you
never get clones in that assay, just a mixed population of independently transfected cells. So you would
be constrained to sequence each candidate first while it was still in E. coli, and then test them one at a
time in independent transfection experiments.
A true shuttle vector would maintain the mutants in the eucaryotic cell long enough to be separated
into individual clones and then subjected to a genetic test or screen or selection. The ones that pass should
then be able to be shuttled back into E. coli for sequencing. True shuttle vectors exist for yeast. Some
vectors exist for mammalian cells that approach these capabilities, but all of them have problems. One
common problem is that reliable tightly controlled promoters are not available, such that you can keep the
transgene turned off until you are ready to do the screen. This leads to the risk of inadvertently conducting
a selection during clonal expansion based on toxicity of the transgene. A second problem is that many of
the vectors integrate into the genome. This allows for multiple integrations that will not segregate apart
the way that plasmids do in bacteria.
Two technical advances exist that may eventually substitute for the shuttle vector in a eucaryotic
saturation mutagenesis experiment. One is the Fluorescence Activated Cell Sorter (FACS) that could
conduct a screen one cell at a time, removing the requirement to grow the transfected population up as
colonies. The second is PCR which may allow recovery of the molecules from individual sorted cells.
Study problem
1. How could you carry out saturation mutagenesis of the SV-40 enhancer?
2. How could you use the fact that human ras will complement yeast ras to carry out saturation
mutagenesis looking for other mutations (besides val 119) that produce an activated ras? (Yeast ras
functions in a pathway that signals vegetative growth under conditions of good nutrition and sporulation
under conditions of poor nutrition).
3. What would you need to recover a collection of stability mutants in rhodanese by saturation
mutagenesis? (Rhodanese transfers a sulfur to cyanide).
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