Download Genetic Techniques for Biological Research Chapter11

Genetic Techniques for Biological Research
Corinne A. Michels
Copyright q 2002 John Wiley & Sons, Ltd
ISBNs: 0-471-89921-6 (Hardback); 0-470-84662-3 (Electronic)
l l Advanced Concepts in Molecular
Genetic Analysis
REVERSE GENETICS
With the advent of recombinant DNA technology and the development of methods
for the purification and sequencing of proteins, it became possible to clone the gene
encoding any purified protein. With the cloned gene in hand, one canuse any one of
a number of techniques to introduce random mutations into a cloned sequence, or
one can induce mutations in regions of interest, such as sites of putative functional
motifs, using a variety of in vitro techniques. The mutant alleles are then tested in
vivo fora change in phenotype. This approach is called reverse genetics. Inthe
classic genetic approach one startswith a phenotype of interest and isolates mutants
with an altered phenotype. No prior mechanistic understanding of the phenomena
controllingthephenotype is required and a large array ofgenesislikely
to be
identified. The classic genetic approach casts a wide net and is capable of providing
abroad viewof a complex biological system. Inthe reverse genetic approach
mutations are directed to a particular gene (protein) and the effects of these changes
on the phenotype of the organism are then determined. Some of these phenotypic
effects can be predicted based on the biochemical and/or biological information
available about this protein. Some of thephenotypes
might be novel and
unexpected. Whatever the outcome, the reverse genetic approach provides detailed
information regarding the function of a particular gene product.
Several methods of in vitro mutagenesis are available to the researcher. Some
induce random changes; others are site directed and can induce specific changes in
particular basepairs or short sequence regions. One should just be aware that there
is tremendous flexibility in the type of mutation that can be induced. Protocols for
these methods are available in the literature and are included with commercially
available kits. These will not be discussed here.
Two very importantconsiderationsmust
be kept in mind when developing a
strategy for testing the invivo phenotype of the mutant alleles. First, mutations
generated in vitro when tested in vivo may not have a detectable phenotype. If they
do have a phenotype, the mutant alleles could be recessive. Therefore, analysis of
the phenotype must be done in strains that carry only recessive mutant alleles of the
gene understudy. Preferably, the other copies of the gene present in thestrain
should be null alleles. This avoids any possibility of an interaction with the mutant
gene product being tested. Construction of a null allele in Saccharomyces is
extremely straightforward using the one-step gene replacement methods described in
Chapter 1. Second, because subtle phenotypic variation among the mutant alleles is
possible, it is importantto test each mutant allele in isogenic strains. This is
necessary in order to be able clearly to associate the phenotype of the mutant with
that specific mutational alteration and not variations in the genetic background.
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The need to use a null allele when testing thephenotype of amutant gene
becomes a problem if the gene product has an essential function. In Saccharomyces
this difficulty is addressedquite simply, butinotherorganismsthispresentsa
greater difficulty. To test whether a gene is essential in Saccharomyces, a null allele
is constructed in a diploid strain. Tetrad analysis of this heterozygous diploid will
give two viable and two nonviable spores if the gene is essential. If the diploid is
transformed with an extrachromosomal plasmid carrying the wild-type allele of the
gene of interest, all four spores should be viable. The two spores containing the
chromosomal null allele are able to grow because of the plasmid copy of the gene
and viability of these spores will be dependentuponcontinued
presence of the
plasmid.
To determinethephenotype
of amutant alleleof the gene of interest,the
heterozygous GENllgenlA diploid is transformed with theextrachromosomal
plasmid carrying the mutant genl gene, sporulated under selective conditions to
maintaintheplasmid,andtetrad
analysis done. If thetwospores with the null
mutation are notviable, then the plasmid-borne mutant allele is not functional. But,
if all four spores are viable, then the mutant allele is capable of providing function,
i.e. it is complementingthechromosomal
null allele. Thefunction of the genl
mutant allele may not be entirely wild type and this can be determined by a detailed
analysis of the phenotype of the mutant spores.
Anothermethod is called the plasmid shuffle. Using theprocedures described
above, a strain containing the chromosomal
null allele and a plasmid-borne wildtype allele (plasmid 1) is constructed. A second plasmid carrying the mutant allele
of the gene and a different nutritional marker for selection (plasmid 2) is introduced into the same host
cell. The doubly transformed host cellis grown under
conditionsthat select for themaintenance of plasmid 2 butnot plasmid 1. If
plasmid 1 can be lost,thenthe mutant gene on plasmid 2 is functional and its
phenotype, if any, can be studied. If the plasmid is never lost despite growth in
nonselective conditions,thenthe
mutant gene on plasmid 2 is nonfunctional.A
very simple way of testing the ability to lose a plasmid uses the pink :white color
change of ade2 versus ADE2 strains, respectively. This is called a colony sectioning
assay. If the selective marker gene on plasmid 1 is ADE2 and the host strain is ade2,
thenthecolonyformed
by this strain willbe white so longasthe cells contain
plasmid 1. If the colony is allowed to grow on a medium containing adenine, then
plasmid 1 can be spontaneously lost so long as the transformant is not dependent
on the wild-type gene also carried by this plasmid. When plasmid 1 is lost the cell
will be ade2, will producethe pink pigment, and all the progeny willbe pink
formingapinksector
in the white colony. Pink sectors are easily observed in
colonies. The researcher can simply scan a large number of colonies growing on an
adenine-containing medium for sectoring. If this is observed, the host cell is not
dependent on plasmid 1 and the mutant gene on plasmid 2 is functional.
A thirdmethod uses tightly regulated promoters, such as C T R l , tocontrol
expression of the wild-type allele carried by plasmid 1 (Labbe & Thiele, 1999). If the
mutant allele carried by plasmid 2 is not functional, then viability of the double
transformant will be dependent on the expression of the wild-type gene, which can
be determined by comparing growth under conditions where the gene is expressed
and not expressed.
ADVANCED CONCEPTS IN MOLECULAR GENETIC ANALYSIS
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COLD-SENSITIVE CONDITIONAL MUTATIONS
Cold-sensitivemutationsare
especially useful because previous experience has
demonstratedthat cold-sensitive mutationsoccurmostoften
in genes encoding
protein components of complex multimeric structures such as ribosomes, kinetochores, viral coats, and other large multiprotein
complexes. Cold sensitivity often
appears to be the result of a decreased ability of the components to assemble into
functional structures at the lower temperature, while at normal growth temperature
assembly occurs atadequaterates.Thus,
if one were interested in isolating
mutations in genes encodingcomponents of suchstructuresorinvestigatingthe
assembly process,oneshouldconsiderisolating
cold-sensitive mutations. This is
done using variousmethods of randommutagenesisonthe
gene carriedona
plasmid vector. The mutagenized pool is transformed into a null mutant host strain
and screened for a cold-sensitive phenotype. If the null
allele is lethal, a method
such as plasmid shuffle would have to be used.
As with temperature-sensitivemutations, cold-sensitive mutations are non-null
mutations. Therefore, if one obtains allele-specific suppressors of a cold-sensitive
mutation, this suggests a physical interaction between the protein with the coldsensitive alteration and the suppressor gene product. If a cold-sensitive mutation
weakens theinteraction at thenonpermissivetemperature,thenthesuppressor
mutation is likely to strengthen the interaction at the nonpermissive temperature. It
is impossible to predict the phenotype of the suppressor mutation in the absence of
the original cold-sensitive mutation. If the interaction with the wild-type product is
unaffected, then the suppressor mutation will have no mutant phenotype. On the
other hand, if the interaction is disrupted, the suppressor mutation might have a
mutant phenotype similar to the original mutation; or, if the interaction is strengthened,thesuppressormutationmighthavea
novel phenotype.Thisinformation
would have to be empirically determined.
DOMINANT NEGATIVE MUTATIONS
Widespread use of dominant negativemutant alleles became possible with the advent
of gene cloningand DNA technology(Herskowitz, 1987). A dominant negative
mutation is one that disrupts the function of the wild-type allele. Thus, the use of
dominant negative mutations has become a very powerful technique for studies of
protein function in organisms where the usual methods of producing mutant alleles
are difficult or impossible. The dominant negative is a way of eliminating the function of a particular gene without having to isolate loss-of-function mutationsin that
gene. One often sees this method used in studies with mammalian cells.
The term dominant negative seems contradictory because a negative mutation,
which implies loss-of-function, should be recessive not dominant. Most often, the
dominant negative mutation requires overexpression to be dominant but occasionally examples arediscovered where this is not necessary. The need for overexpression
is explained as follows. Many proteins bind to other proteins and form multiprotein
complexes in which only one component has the catalytic activity while the others
function as regulatorsof the catalytic subunit.A mutation in the catalytic domainof
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the protein will affect its catalytic activity but will not necessarily affect its binding to
the regulatory components in the complex. If the other components are made
in
limiting amounts, then the overproduced mutant subunit will bind to and sequester
(titrate) the available subunits and make them unavailable to the small amount of
wild-type protein synthesized by the otherwise wild-type host cell. If the limiting
regulatory subunit is a positive regulator a null-like phenotype will be produced. If
the limiting subunit is a negative regulator a constitutive phenotypewill be produced.
In this manner, a loss-of-function mutation can dominate the phenotype of a wildtype host.
The Cdc28 protein is a cyclin-dependent protein kinase and binding of proteins
called cyclins is necessary to activate the kinase activity of Cdc28p. Cdc28p kinase is
an essential protein needed for the progress from GI to S and G2 to M in the cell
cycle. Cyclin proteins are present in limiting amounts at certain times in the cell
cycle and if these are depleted the cell will be unable to enter the S phase or mitosis.
If a mutation is introduced into CDC28 at a site encoding an essential residue of the
kinase activity, such as in the ATP binding site, then the encoded protein willbe
inactive as a kinase and the mutation will be recessive. However, the ability of the
altered Cdc28p to bind cyclin has not been affected by this mutation. Therefore, if
this mutant cdc28 gene is overexpressed, the mutant protein product will be very
abundant and the cyclin proteins will bind to this nonfunctional protein instead of
to the much less abundant wild-type Cdc28p encoded by the chromosomal copy. By
so doing, the mutant product blocks the activation of the wild-type Cdc28 protein
thereby preventing it from functioning. Thus, when this recessive cdc28 mutation is
overexpressed the nonfunctional phenotype becomes ‘dominant’. In this way, the
geneticist can explore the phenotype of a cdc28 null mutation without having to
construct one. This is extremely useful in mammalian cells where constructing a
deletion mutation is an extremely lengthy and expensive undertaking.
Occasionally, one finds a dominant loss-of-function mutation that does not need
to be overexpressed. In these cases the gene product is part of a multiprotein complex
of like subunits and the mutationhas altered a functional activity of the protein but
not its ability to form the multiprotein complex. The dominance results from the fact
that even if one subunitof the complex is mutant theentire complex is nonfunctional.
The tumor suppressor protein p53 is a noteworthy example (KO & Prives, 1996).
The dominant negative mutation has additional uses. For example, one can use it
to isolate the gene encoding the interacting protein
using multicopy suppression.
The mutant phenotype produced by the overexpression of adominant negative
mutant gene should be suppressed by the overexpression of the interacting protein.
One would use some type of overexpression library, introduce this into the host cell
carrying the overexpressed dominant negative mutation, and select/screen for wildtype-like suppressor-containing transformants. The suppressing plasmid can easily
be recovered and the gene responsible for the suppression identified.
The dominant negative strategy can be used for structure-function analysis of the
catalyticsubunit of amultiprotein enzyme complex. It also could be used to
characterize a multiprotein DNA-binding complex. Mutation of the DNA-binding
domain but not the domains
used for protein-protein binding should produce a
dominant negative allele. Similar approaches could beused for studies of other
multiprotein complexes.
ADVANCED CONCEPTS IN MOLECULAR GENETIC ANALYSIS
111
CHARGED-CLUSTER TO ALANINE SCANNING
MUTAGENESIS
Charged-cluster to alanine scanning mutagenesis
is a ‘semi-random’ approach to
choosing which residues to mutate so as to improve the probability of affecting the
function in ameaningful way. Alterations that causethe gene productto be
unstableareuninterestingand
do not allow one to explorefunction.Chargedcluster to alanine scanning mutagenesisis a method that optimizes the generationof
mutant alleles with stable gene products.
X-ray crystallography has been used successfully to reveal the three-dimensional
structure of many proteins. Generally speaking, clustersof charged residues tend to
be located at the surface of a protein while hydrophobic and nonpolar residues are
often buried in the core of the protein. The charged-cluster to alanine approach to
constructing in vitro mutations uses this finding to propose the following. First, if
one scans a protein sequence those regions containing clusters of charged residues
have areasonableprobability
of being positioned at thesurface of thefolded
protein. Second, alterations of surface residues should produce fewer of the types of
structural abnormality that often make mutant proteins the target of proteolytic
degradation.Third,changes in these chargedclustersare likely to altersurface
residues that are often involved in protein-protein interactions. These predictions
have proven to be correct frequently enough to make charged-cluster
to alanine
mutagenesisavaluabletooland
widely acceptedasamethodfor
genetically
dissecting the different protein-protein interactions of a particular multifunctional
protein or protein component of a multiprotein complex. The types of interaction
that can be detected by this approach are interactions with substrates, activating
subunits,inhibitorysubunits,targetingsubunits,andothercomponents
of complexes. For proteins that function in several processes, the charged-cluster to alanine
scanning approach is able to dissect these different functions because the clustered
alterations may affect only one of the several functions.
One scans the sequence of a protein usually using an overlapping window of five
residues looking for the presence of two or more charged residues in the window.
All thecharged residues are thenchanged to alanine using in vitro mutagenesis
techniques. The mutant allele is then tested for phenotype in a null mutant strain as
described above in the section on reverse genetics.
REFERENCES AND FURTHER READING
Bass, S.H., M.G. Mulkerrin, & J.A. Wells (1991) A systematic mutational analysis of
hormone-binding determinants in the human growth hormone receptor. Proc. Natl Acad.
Sci. USA 88: 4498-4502.
Bennett, W.F., N.F. Paoni, B.A. Keyt, D. Botstein, A.J. Jones, L. Presta, F.M. Wurm, &
M.J. Zoller (1991) High resolution analysis of functional determinants on human tissuetype plasminogen activator. J. Biol. Chem. 266: 5191-5201.
Cunningham, B.C. & J.A. Wells (1989) High-resolution epitope mapping of hGH-receptor
interactions by alanine-scanning mutagenesis. Science 244: 1081-1085.
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Herskowitz, I. (1987) Functionalinactivation of genes by dominant negative mutations.
Nature 329: 219-222.
KO, L.J. & C. Prives (1996) p53: Puzzle and paradigm. Genes Dev. 10: 1054-1074.
Labbe, S. & D.J. Thiele (1999) Copper ion inducible and repressible promoter systems in
yeast. Meth. Enzymol. 306: 145-153.