Download 5 Complementation Analysis: How Many Genes are Involved?

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)
5 Complementation Analysis: How Many
Genes are Involved?
Complementation analysis is used to determine whether two independent mutations
arealterations in the same gene; that is, they are alleles, orarealterations
in
different genes. In essence, a complementation analysis is a functional test used to
define a gene. If a researcher has isolated anumber of mutants with a similar
phenotype, the next question asked is: ‘How many genes have I identified?’. If there
are 10 mutant strains, are they each in different genes, does each mutant carry a
different mutation (allele) in the same gene, or something in between such as two
genes one with six alleles and the otherwith four alleles? Complementation analysis
will help answer this question.
Seymour Benzer’s study of the rZZ locus of phage T4 is a most elegant example of
the power of complementation analysis (Benzer, 1955). Benzer had several hundred
mutations that gave the same phenotype, large plaques on one host strain of E. coli
and no plaques on another host strain, and mapped to the same region of the T4
chromosome. Using the host strain in which r l l mutants formed no plaques, he
found that when host cells were coinfected with different mutant pairs some pairs
produced a normal phage burst while others did not. Those that produced a normal
burst he concluded were in different functional genetic units that he called cistrons, a
term that is synonymous with gene. In contrast, those pairsof mutants that rarely or
never produced a normal burst
he concluded were in the same cistron. The rare
productive infections Benzer proposed resulted fromrecombination between the
different mutations in the same cistron thereby creating a wild-type genotype in a
few coinfected cells. Using this method, he placed all of his r l l mutations into two
cistrons that he called r l l A and rllB. Moreover, he made a detailed genetic map
based on recombination frequency between the different mutations (a fine structure
map) that also indicated the frequency at which a mutation was isolated at that
position. In this way he demonstrated that genes are not indivisible units but consist
of many mutable sites that can recombine. Geneticists working with other
organisms soon followed Benzer’s lead and adapted complementation analysis to
their systems.
To carry out a complementation analysis, both mutant genes must be expressed in
the same cell so that their gene products are synthesized in the same cytoplasm and
can functionally interact. Only loss of function (recessive) mutations can be used for
acomplementation analysis. Thetheory behind thecomplementation analysis is
simple. If both mutations are loss of function alterations of the same gene, then the
diploid cell carrying these two mutant genes will not contain a functional allele and
will have the mutant phenotype. If both mutations are loss of function mutations
but in different genes, then the diploid cellwill have one mutant allele and one
functional wild-type alleleof each gene and, since the mutant alleles are loss of
function alleles, the diploid will have the wild-type phenotype.
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To express both mutant genes in the same cytoplasm a heterozygous diploid must
be constructed. The way the researcher establishes the diploid state varies with the
organism under study. In Saccharomyces, this is accomplished by mating a MATa
strain containing mutation #l to a M A T a strain containing mutation #2. The ala
diploid will be heterozygous for the mutant genes. The phenotype of the heterozygous diploid is then observed. If the diploid has a wild-type phenotype, then the
mutations are said to complement and this is strong evidence that the mutations
are in different genes. A geneticist might also say, ‘The mutations are in different
complementation groups’. If the diploid has a mutant phenotype, then the mutations
do not complement and are said to be in the same complementation group. This is
considered strong evidence that the mutations are alleles. The definition of complementation group is a set of noncomplementing mutations. The term complementation group is synonymous with gene.
Two conditions must bemet before one can carryout a complementation analysis
on a series of mutants. First, the mutant strain can only contain a single mutation
compared with the parental strain. Particularly when mutagenesis had been used to
obtain the mutants, it is possible that more than oneDNA alteration was induced in
an individual and these are involved in producing the mutant phenotype. Therefore,
each mutant strain must be tested todemonstrate whether one or more genetic
alterations are required to produce the mutant phenotype. To test this the mutant
strain is crossed to a wild-type strain and tetrad analysis of the heterozygous diploid
is carried out. If only a single alteration is required, then only tetrads with two
mutant spores and two wild-type spores will be produced, as shown in Cross 1 in
Chapter 1. But if two or more alterations are present, tetratype and nonparental
ditype tetrads will be produced, as shown in Cross 3 in Chapter 1. What would be
the phenotypes of the spores of a tetratype tetrad if the mutant strain containedtwo
altered genes and both alterations were required to produce the mutant phenotype?
What would be the phenotypes of the spores of a tetratype tetrad if the mutant
straincontained two altered genes and eithermutationalone
were sufficient to
produce the mutant phenotype?
Of course to carry out a cross between themutantand
wild-type strainsthe
strainsmustbe
of oppositematingtypeandshouldcarry
different nutritional
mutationsto facilitatethe selection of diploids. But in other respects the two
strains should ideally be isogenic except for any alterations required to produce the
mutant phenotype. Usually, before undertaking a mutant hunt, the geneticist will
construct an appropriate pair of isogenic (or congenic) haploid strains to be used
as parental strains. The mutants isolated in one strain can then
be mated to the
parental strain of the opposite mating type to determine the number of mutant
genes involved.
As described above, the second requirement for a complementation analysis is
that the mutations be loss of function alleles. In other words, only mutations that
are recessive to the wild-type allele can be used. So, as a second step in the genetic
analysis of mutants, mutant strains carrying a single mutant gene are crossed to a
parental strain carrying the wild-type allele. If the mutant carries a recessive loss of
functionmutation,thenthe
heterozygous diploid (GENllgenl-34) will have the
wild-type phenotype.Thismutant
allele canthen be used forcomplementation
analysis.
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Cross 4 shows a complementation test for two mutant strains. Preliminary genetic
analysis has shown that each strain contains only a single mutant gene and that the
mutant allele is recessive.
Cross 4:
Diploid
phenotype:
Mutant strain 5
Mutant
x
Mutant strain 14
The result shown in Cross 4 indicates that the mutationin strain 5 and the mutation
in strain 14 do not complement and thus are mutations in the same gene. If we call
the gene GENI, then these mutations are alleles and one could now name them
genl-5 and genl-14. This cross could be depicted as shown below.
Cross 4:
genl-5
X
genl-l4
(mutant)
(mutant)
(phenotype
Diploid:
genl-5
(genotypes of parental
strains)
of parental
strains)
(genotype of diploid)
genl -I 4
(mutant)
(phenotype
of diploid)
As a second test of whether or not the mutations are alleles, the researcher can
determinethe segregation pattern of the allelesin the meiotic products of the
diploid. If the two mutations are in the same gene, then recombination between
the mutations will be relatively rare because they map so close to one another.
Therefore, 100% of the time (or close to it) the two mutant genes will segregate to
different spores producing a tetrad with four mutant spores (two mutant # 5 spores
and two mutant #14 spores). This situation is depicted in Cross 2 of Chapter 1).
Cross 5 shows anothercomplementation test between mutantstrain 5, which
carries the mutation genl-5, and another mutant strain. Mutant strain 4 contains
only a single mutant gene and the mutant allele is recessive.
Cross 5 :
Diploid
phenotype:
Mutant strain 5
Wild-type
x
Mutant strain
4
The result shown in Cross 5 indicates that the mutation in strain 5 and the mutation
in strain 4 complement and thus are mutations in different genes. We can then say
that a different gene, GEN2, is mutant in strain 4. This cross could be depicted as
shown below.
Cross 5:
genl-5
GEN2
(mutant)
(mutant)
(phenotype
Diploid:
genl-5
gen2-4
-GENl GEN2
(wild-type)
X
GENl gen2-4
(genotypes of parental
strains)
of parental
strains)
(genotype of diploid)
(phenotype of diploid)
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TECHNIQUES
BIOLOGICAL
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If GENl and GEN2 are not linked, then the mutant genes will recombine producing recombinant meiotic products with the wild-type (GENI GEN2) and double
mutant (genl-5 gen2-4) genotype. This is exhibited by the presence of tetratype and
nonparentalditypetetrads
when this diploid is subjected to tetrad analysis (see
Cross 3 in Chapter 1). The frequency of each type of tetrad will depend on the
frequency of recombination. If the two genes are completely unlinked, that is 50%
recombination, the frequency of PD : TT : NPD tetrads will be 1 : 4 : 1. If there is any
linkage, thenthe frequency of recombination isless than 50% and the relative
number of PD tetrads will increase to greater than the
expected 116 of the total
number of tetrads analyzed. Ultimately, for crosses between two alleles, the number
of P D tetrads will closely approach loo%, as is shown in Cross 1. One can calculate
the map distance between two mutations (the frequency recombination multiplied
by 100) using the following formula, which is correct for map distances up to 35 CM
(Sherman & Wakem, 1991):
map distance in CM =
+
~
2
6 x # NPD tetrads # TT tetrads
total # of tetrads
Thecombination of these twomethods,complementation
analysis and tetrad
analysis, should clearly indicate whether one is dealing with mutations in one or
more genes. Either method alone is not as powerful, and therefore researchers do
both tests. For example, if there are mutations in two very tightly linked genes, the
mutations will complement, but recombination will be rare and most, if not all the
tetrads will be PD. Such results would strongly suggest that one is dealing with
mutations in two closely linked genes.
In a complete complementation analysis, all the mutants are crossed to all of the
other mutants. Often as a complementation group containing several mutant alleles
is identified, one allele will be chosen as the representative of that complementation
group and only this allele will be crossed to the other mutants. At the end of this
process, all the mutants isolated in aparticularmutant
selection/screen will be
placed into complementation groups, i.e. genes. The researcher will have made a
good start at determining the number of genes involved in the process of interest. If
only a few genes have been identified with several mutant alleles, then the researcher
will have some degree of confidence that the analysis saturated the genome and that
new genes are not likely to be identified by the same selection/screening method. If
many genes have been identified, several with only one mutant allele each, then it is
likely that new genes will be identified if the same selection/screen is repeated.
There are some special situations in which straightforwardinterpretation of a
complementation test is misleading and the geneticist must be on the alert for such
possibilities. Infrequently, mutant alleles of the same gene are able to complement,
and produce a heterozygous diploidwith a wild-type like phenotype. This is referred
toas intragenic complementation. Intrageniccomplementationcan
occur if the
encoded polypeptide forms a multiple subunit protein composed of like subunits,
such as a homodimer, or if it encodes a single polypeptide that carries out several
distinctfunctions.Inthe
case of thehomomultimericprotein,
mutant subunits
encoded by different mutant genes associate with one another in the multimeric
protein and are able to accommodate each other’s mutant alteration in the mixed
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COMPLEMENTATION
multimer. When this happensthemixed-mutant
multimer complex has some
functional activity, although it may not be completely normal.
Another mechanism of intrageniccomplementation is possible if theprotein
product of the gene has several distinct functions, such as two different enzyme
activities. In this situation it is possible to obtain mutations that affect one of these
activities while leaving the other function intact. In a heterozygous diploid, cells
carrying two different mutations, each one affecting only one of the two functions,
proteins capable of carrying out both enzyme activities will be produced, albeit in
different molecules, and the cell should have the wild-type phenotype.
In contrast to intrageniccomplementation where mutations in the same gene
complement, in a few instances mutations in different genes which are expected to
complement do not. This phenomenon is referred to as nonallelic noncomplementation. One explanation for this noncomplementation
is that the two genes encode
subunits of a heteromultimeric protein, and that the presence of a mutant alteration
in either subunit destroys all function of the multimeric protein. Sort of, ‘one bad
apple spoils the whole barrel’.
Careful and thorough genetic analysis involving both complementation tests and
genetic mapping of several mutant alleles is necessary to avoid the pitfalls of these
potentially misleading situations.
REFERENCES
Benzer, S. (1995) Fine structure of a genetic region in bacteriophage.
USA 41: 344-354.
Sherman, F. & P. Waken (1991) Mapping yeast genes.
Proc. Natl Acad. Sci.
Methods Enzymol. 194: 38-57.