Download 4B. Complementation

4B. Complementation
Complementation analysis examines the phenotypic effect of having two or more alleles present in the
same organism. In concrete terms, you are comparing situations in which there are multiple forms of
one gene product or of several different gene products in the same cell. The abstract interactions
among the alleles constrain how the gene products interact.
By constructing organisms that are heterozygous for each of two recessive mutations one can test the
functional relationship of those mutations. If the two mutations are in the same functional unit, then
the heterozygote (a1 +/+ a2) will have the mutant phenotype. If the two mutations are in different
functional units, then the heterozygote (a1/+ b1/+) will display the wild-type phenotype.
Complementation tests define functional units
Cis and trans tests of recessive alleles allows inference of whether those alleles affect one or more
“functional units” also known as cistrons or “genes.”
Complementation tests are done with respect to a given phenotype. Two alleles can complement for
one phenotype but fail to complement for another phenotype.
Constructing trans-heterozygotes.
Linked markers can be used to identify the relevant heterozygote.
Consider recessive marker m conferring phenotype M, and alleles a and b to be tested for
complementation.
m a / + + male x m b / m b female 
½M
ma/mb
Score phenotype
½ non-M
++/mb
Of course there is some degree of recombination in the heterozygous parent. Recombiantion
would yield m + and + a gametes at a frequency of p, the map distance between the m and a loci.
A dominant mutation (or transgene) can be used to keep track of the other chromosome.
Insertion of a GFP or of an RFP would indicate the genotypes.
a + / + GFP male x b / b female 
½ Gfp
½ non-Gfp
+ GFP / b
a+/b
Score phenotype
With two dominant balancers, both parents can be heterozygous.
a / GFP male x b / RFP female 
2011 P.Sternberg & L Huang ch. 4B ¼ Gfp non-Rfp
¼ Gfp Rfp
¼ non-Gfp Rfp
¼ non-Gfp non-Rfp
4/18/11 GFP / b
GFP / RFP
a / RFP
a / b Score phenotype
1
As with the linked marker, recombination could be an issue, and thus
Balancer chromosomes can be used where available. Balancer chromosomes include a dominant
marker and a cross-over suppressor, for example a rearrangement that prevents pairing (multiple
inversions or in some organisms, reciprocal translocations.)
These work just like the dominant markers in trans described above but there will be no
recombination.
In yeast, one can simply mate two haploid strains and score the diploid.
MATa a
x MATα b  MATa/MATα a/b diploid
Genotyping individuals. In the worst case, individuals can be genotyped by molecular markers.
This can be for linked SNPs, or the alleles a and b if their molecular basis is known. For example, a
small amount of tissue (blood from mouse tail) can be examined.
Exceptions to complementation are highly informative:
If two alleles fail to complement but map at distinct loci (e.g., different chromosomes) this is
considered “extragenic non-complementation” or “non-allelic non-complementation.” It implies some
functional connection between the two genes defined by those two alleles.
If two alleles of the same gene complement, this is considered “intragenic complementation” and is
indicative of either interaction among the gene products or multiple independent functions of the gene
or its product(s). (You know they are allelic by fine-structure mapping, DNA sequence, or by their
failure to complement another class of alleles).
Complementation in transgenic organisms can connect DNA to alleles.
Genotype
gene A
DNA
+/+
none
+/none
-/none
-/A(+)
-/A(-)
2011 P.Sternberg & L Huang ch. 4B Phenotype
non-A
non-A
A
non-A
A
4/18/11 2
gf and dn mutations can identify phenotypes for a multigene family.
Imagine there is a family of genes that encode identical or essentially identical proteins that are
expressed in the same places. Inactivation of a single gene might have no discernible phenotypic
effect (but might confer some selective disadvantage.) However, a gain-of-function might well cause a
phenotype. The challenge becomes sorting out whether the gain-of-function effect is indicative of the
genes normal function, that is, whether it is a hypermorph. A dominant negative can also indicate
normal function.
RNAi cross-target effects can be an advantage in knocking down function of a gene family.
A series of alleles can reveal how gene activity can vary.
An allelic series is a set of alleles that display a graded effects on penetrance or expressivity. A
simple case of an allelic series are alleles that affect the level of enzyme activity encoded by the gene.
Complex complementation indicates genes with multiple functions
Some alleles have pleiotropic effects, that is, they have multiple phenotypes. Some disparate
phenotypes might arise from indirect effects. For example, a problem in food intake might affect
locomotion, longevity, etc. It is thus important to define the primary defect(s). In other cases, the
gene is acting at multiple times and places. The time and spatial location of gene action will be
discussed in section 4C, but complementation analysis can reveal multiple roles of a gene.
Compensating mutations can underlie intragenic complementation
Salt bridges in protein (between oppositely charged side-chains) are one of the best cases of
compensating mutations.
genotype
.…K….…D….
.…D….…D….
.…K….…K….
.…D….…K….
Table/Figure.
phenotype
/ .…K….…D….
wild-type
/ .…D….…D….
mutant
/ .…K….…K….
mutant
/ .…D….…K….
wild-type
K, positively charged lysine residue; D, negatively charged aspartic acid residue.
Distinct places or times of expression can underlie intragenic complementation
Table -. different expression domains
Genotype
+/+
h 1/ h 1
h1/+
h 2/ h 2
h2 /+
h 1/ h 2
Phenotype
Tail
+
+
+
+
+
Belly
+
+
+
+
+
Distinct functional domains of multidomain proteins can be identified by intragenic
complementation
The epidermal growth factor receptor (EGF-receptor) has two major domains separated by a singlespan transmembrane domain. An extracellular domain binds ligand (EGF), while an intracellular
domain transduces the signal. The receptors dimerize upon lgand binding thus allowing the
intracellular kinase domain of one molecule to phosphorylate its partner on tyrosine residues. Other
2011 P.Sternberg & L Huang ch. 4B 4/18/11 3
proteins, containing SH2 domains, bind peptides containing phosphotyrosine and lead to downstream
signal transduction, as well as regulation of signal transduction.
genotype
ligand-binding+
ligand-bindingligand-bindingligand-binding+
ligand-binding+
ligand-bindingligand-bindingligand-binding+
kinase+ / ligand-binding+ kinase+
kinase- / ligand-binding- kinasekinase- / ligand-binding+ kinase+
kinase- / ligand-binding+ kinasekinase- / ligand-binding+ kinase+
kinase+ / ligand-binding- kinase+
kinase+ / ligand-binding+ kinase+
kinase- / ligand-binding- kinase+
activity
+
+
(+)
(+)
+
Distinct molecular interactions can underlie intragenic complementation.
In contrast to proteins with multiple domains, calmodulin has one domain but nonetheless displays
intragenic complementation.
Of a series of Phe to Ala mutations in the Yeast CMD1, 8 mutations had defects and fell into four
groups
group
A
B
C
D
F16A, F19A
F89A, F140A
phenotype
actin organization
Cmd1p mislocalization to bud
nuclear division
bud emergence
2011 P.Sternberg & L Huang ch. 4B 4/18/11 4
http://www.rcsb.org/pdb/molecules/pdb44_2.html
“One common theme in the contact between calmodulin and its different target proteins is the use of
non-polar interactions, in particular, through the interactions with the unusually abundant methionines
of calmodulin. Calcium binding exposes these non-polar surfaces of calmodulin, which then bind to
non-polar regions on the target proteins. The structure shown on the left, from PDB entry 1cfd, shows
calmodulin without calcium, and the structure on the right, from PDB entry 1cll, shows calmodulin after
calcium binds. The key nonpolar areas are colored with carbon atoms in green and the many
methionine sulfur atoms in yellow. Notice how these non-polar amino acids form two neat grooves
(shown with red stars) when calcium binds, waiting to grip the target protein. Because these non-polar
grooves are generic in shape, calmodulin acts as a versatile regulatory protein and its targets are not
required to possess any specific amino acid sequence or structural binding motifs”
Multiple Function Hypothesis
Genotype
Phenotype
1
2
a1/a1
+
a2/a2
+
anull/anull
a1/a2
+
+
a1/anull
+
a2/anull
+
-
3
-
Level Hypothesis
Genotype
Phenotype
1
2
a1/a1
+
a2/a2
anull/anull
a1/a2
+/a1/anull
+/a2/anull
-
3
+
+
+
+/+/-
2011 P.Sternberg & L Huang ch. 4B 4/18/11 5
Extragenic non-complementation
Simultaneous reduction of two products results in a phenotypic abnormality.
Not allele specific. Does not imply interaction.
Two mutant forms interact to form a poisonous product
Allele-specific at both loci. Implies interaction.
One mutant sequesters the wild-type form of other product into an inactive complex
Allele-specific at one locus. Implies interaction.
2011 P.Sternberg & L Huang ch. 4B 4/18/11 6