Download Genetic analysis of mutation types

Phenotype and Function
Reading 222-224
Problem set D
Genetics can be used as a tool to study problems in biology and has
made important contributions to the fields of development, cell
biology and neurobiology. One of the goals in this type of research
is to use mutations to reveal the normal function of a gene.
However, to infer the function of a gene from mutant phenotypes,
we need to know how the mutation perturbs the activity of the
gene. Two questions are particularly important to address. (1)
Does a recessive mutation reduce or eliminate gene function? (2)
Does a dominant mutation reduce or alter gene function?
Recessive or dominant?
The first goal in understanding the nature of a mutation is to
determine if it is dominant or recessive. In most cases mutant alleles
are recessive to the wild-type alleles. There are also more
complicated cases where two alleles are neither dominant nor
recessive. If the heterozygote expresses the phenotype of both
alleles, the alleles are called codominant. This is most frequently seen
with molecular assays for gene function, for example, in ABO blood
types. The phenotype of the heterozygote can also be intermediate
between the phenotypes of the homozygotes. In these cases the
alleles are referred to as incompletely dominant.
Loss-of-function mutations
Recessive mutations almost always reduce or eliminate gene activity.
Mutations that reduce gene function are called weak or
hypomorphic. Mutations that eliminate gene function are called null
or amorphic. Whether a mutation is hypomorphic or amorphic can
be determined by genetic criteria.
Hypomorphic mutations reduce but do not eliminate gene
activity. An example is the apricot allele (wa) of the white gene. This
mutation is recessive and results in apricot colored eyes. If we
compare the phenotypes of wild-type and wa alleles, there are two
interpretations of how the white gene functions. One possibility is
that white produces eye color and the wa allele reduces that
production. Alternatively the white locus could inhibit eye color and
the wa allele increases the effectiveness of white to inhibit eye color.
In 1932 Hermann Muller proposed that one could distinguish
between these possibilities by changing the dose of the wa allele. If
white produces eye color then increasing the dose of wa should
increase eye color. However, if white inhibits eye color, then
increasing the dose of wa should lead to less eye color. In fact when
Muller conducted the dosage experiments with wa, he found that as
the dosage of w a increased the mutant phenotype is less severe.
wa/Df(w) > wa/wa > wa/wa/Dp(wa)
more mutant (less pigment)-------------------less mutant (more pigment)
Amorphic mutations eliminate gene activity and behave similar
to a deficiency of the locus. Recessive alleles of white (w) that lead to
a white eye phenotype are considered to be amorphic. As w dosage
increases, the mutant phenotype remains unchanged.
w/Df = w/w = w/w/Dp(w)
more mutant (less pigment)-------------------less mutant (more pigment)
Hypomorphic alleles can cause a range of mutant phenotypes.
The albino gene of mice encodes tyrosinase, which is one of the
enzymes that produces the fur pigment melanin from tyrosine.
Mutant alleles of various strengths have been isolated. Patches of
skin from these mice can be incubated with radioactive tyrosine and
the amount of tyrosine incorporated into melanin can be measured.
The table below is a comparison of the fur color phenotypes and
levels of tyrosinase from mice carrying various albino alleles. C is the
wild-type gene, c is the amorphic allele and the remaining alleles are
hypomorphic.
Genotype
C+/C+
C+/c
cch/cch
ce/ce
c/c
fur
color
Black
Black
grey
off
white
white
cpm of C14-tyrosine
incorporated
1200 +/- 36
617 +/- 33
442 +/- 15
98 +/- 11
47 +/- 5
From both the phenotypes and biochemical assays we can see that
the different alleles have various activities: C+ > cch > ce > c. This
kind of graded set of alleles is called an allelic series.
When inferring the function of a gene, it is important to know
the null phenotype, the phenotype in the absence of gene function.
However, null alleles can often result in phenotypes that are difficult
to interpret, and in these cases hypomorphic alleles can be more
informative. For example, mutations that disrupt signalling through
the Ras pathway in C. elegans cause a lethal phenotype that is not
understood. However, the Ras pathway is also important for cell
signalling during generation of a vulva, which occurs late in
development well after the Ras pathway null mutants die. Because
hypomorphic mutations in these genes result in no or partial lethality,
animals homozygous for hypomorphic mutations can develop into
adults with abnormal vulvae, revealing the role of these genes in cell
signalling.
Dominant mutations: haploinsufficient or gain-of-function?
Haploinsufficient mutations
Dominant mutations can reduce (haloinsufficient alleles) or alter
(Gain-of-function alleles) gene function. Dominant mutations that
cause a reduction or elimination of gene function define genes that
are required in two doses. These mutations are known as
haploinsufficient because the haploid dose of the gene is not
sufficient for normal gene function. Thus, unlike the albino gene
described above in which reducing gene function 50% does not have
a phenotypic consequence, haploinsufficient mutations define genes
in which a 50% reduction in gene function does affect phenotype.
Animals heterozygous for a haploinsufficient mutation (m/+) have a
phenotype similar to animals heterozygous for a deficiency for the
locus (Df/+).
In Drosophila, there are about 50 Minute genes that are defined
by haploinsufficient mutations. These mutations cause the same
phenotype: homozygous flies die, and heterozygous flies have thin
bristles and develop slowly. One of the Minute genes has been
shown to encode a ribosomal protein.
Gain-of function mutations
There are three types of gain-of-function mutations. These
mutations are usually, but not always, dominant to their wild-type
alleles.
Hypermorphic mutations increase gene activity relative to their
wild-type alleles. Adding wild-type gene activity to the
hypermorphic gene activity leads to a more severe mutant
phenotype. One example of hypermorphic alleles are dominant
alleles of the C. elegans gene lin-12 [lin-12(d)]. The lin-12 gene is
required for certain cells to determine their fates normally.
genotype
lin-12(lf) / lin12(lf)
lin-12(+) / lin12(+)
lin-12(gf) / lin12(gf)
Cell A
B fate
Cell B
B fate
A fate
B fate
A fate
A fate
One copy of lin-12(gf) transforms some B cells to the A fate. As the
dosage of wild-type alleles is increased more B cells are transformed
to the A fate.
lin-12(gf)/+/Dp lin-12(+) > lin-12(gf)/+ > lin-12(gf)/Df
more mutant
more B cells transformed to A cells
less mutant
fewer B cells transformed to A cells
Thus lin-12(gf) alleles increase lin-12 gene activity, resulting in the
transformation of B cells to the A fate. lin-12 amorphic and
hypomorphic alleles [lin-12(lf)] lead to cell fate transformations in the
opposite direction, transforming cell A to a B fate. The two types of
lin-12 alleles that cause opposite transformations in cell fate have led
to a model in which the lin-12 gene functions as a binary switch in
cell fate determination: if lin-12 is active cells adopt fate A, if lin-12 is
inactive cells adopt fate B.
2. Antimorphic mutations, which are also called dominant negative
mutations, alter gene activity to antagonize the activity of the wildtype allele. Wild-type gene function depends on the ratio of mutant
gene dosage to wild-type dosage: the higher the ratio, the more
severe the mutant phenotype.
mutant (m)/wild type
(+) ratio
genotype
phenotype
2:1
1:1
1:2
m/m/Dp(+
)
more
mutant
m/+
m/+/Dp(
+)
less
mutant
Examples of antimorphic mutations are certain missense mutations in
the DNA binding domain of the lac repressor. Because the lac
repressor is a tetramer, mutant repressors can complex with wildtype repressors and poison the complex. In this case as the ratio of
mutant:wild-type alleles increases, more defective tetramers are
formed, and the phenotype becomes more mutant.
3. Neomorphic mutations lead to novel gene activity. These alleles
differ from hypermorphic and antimorphic alleles in that they are
insensitive to dosage. Dominant mutations in the Drosophila
Antennapedia (Antp) gene can have a dramatic neomorphic
phenotype. Flies with one copy of the dominant AntpNs mutation
have legs growing out of there heads in place of antennae. AntpNs
mutations are neomorphic because they are insensitive to changes in
the dosage of the wild-type gene
AntpNs / Df = AntpNs / + = AntpNs / + /Dp(+)
The Antp gene is normally transcribed in the thorax where it
promotes leg development. The AntpNs mutation is caused by the
insertion of a transposable element upstream of the Antp gene which
leads to Antp expression in the head. This ectopic expression of the
Antp gene promotes leg formation in the wrong place.
In the next series of lectures, we will discuss how genetics has been
used as a tool to understand a developmental signaling pathway.
What you have learned in this lecture will be used again later on to
determine how mutations disrupt gene function in specific cases.
Problem set D
1. You find a mouse with no tail. In order to determine whether this
mouse carries a new mutation, you cross it to a normal mouse. All
the F1 progeny of this cross are wild type. What does this mean?
You then mate all the F1 males to their sisters and observe that three
out of 42 F2 animals have no tail and two have short tails. What
could explain this pattern of inheritance?
You map the no tail mutation by recombination and realize that there
is a stock available that is heterozygous for a deletion that removes
the region where the no tail mutation maps. You cross animals
without tails to animals that are heterozygous for the deletion. All
the progeny for this cross are wild type. Assuming that the deletion
really removes the gene mutated by your mutation, explain this
result.
2. You are studying the regulation of the lac operon in E. coli and
perform a merodiploid (partial diploids) analysis with various
regulatory and structural gene variants that you isolated. Your first
results are shown below (in units of ß-galactosidase activity).
Experiment
Genotype
1.
2.
3.
4.
lacI+O+Z+
lacI-OcZ+
lacI-OcZlacI-O+Z+/lacI+O+Z+
ß-galactosidase activity
+ Inducer
- Inducer
100
100
1
200
0.1
100
0.1
0.1
lacI encodes the lac repressor and is active in the absence of inducer
(i.e., lac repressor binds to the lac operator and inhibits transcription
from the lac promoter). Addition of inducer inhibits the repressor
and stimulates transcription from the lac promoter. lacO is the
operator, the region to which lac repressor binds, and lacOc
mutations are constitutive operator mutations, causing lacZ
expression in the presence of repressor and absence of inducer. lacZ
encodes ß-galactosidase, an enzyme that functions as a tetramer.
a)
Why is the induced activity in experiment 4 twice that of 1.
You generate additional merodiploids and get the surprising results
shown below.
Experiment
Genotype
5.
6.
7.
8.
lacI-OcZ-/lacI+O+Z+
lacI+OcZ-/lacI-O+Z+
lacI-O+Z-/lacI+OcZ+
lacI+OcZ+/lacI-O+Z-
Induced
Uninduced
10
10
10
10
0.1
0.1
100
100
b) Describe a hypothesis to explain the results of experiments 5-8.
3. Drosophila homozygous for an allele of the thick veins gene, tkvSz2,
are normal-appearing adults except that the veins on the wings are
much thicker than the wild-type wing veins. Flies hemizygous for
that allele and a deficiency of the gene (tkvSz2/Df) die as embryos.
a. What is the nature of the tkvSz2 allele (amorph, hypomorph,
haploinsufficient, hypermorph, antimorph, neomorph)?
b.
Based on the mutant phenotypes, what are the functions of
the wild
type thick veins gene?
It plays an essential role during development of flies and also
functions in regulating the morphology of wing veins.
4. You compare the phenotype of animals that are homozygous for a
mutation (m/m), that are heterozygous for the mutation (m/+), that
are hemizygous for the mutation (m/Df), that contain an extra copy
of the wild-type gene (m/m/+), and that are hemizygous for the
locus (Df/+). Which of these animals will exhibit a wild-type
phenotype, and of the animals that exhibit a mutant phenotype,
which will exhibit the more severe phenotype (order the mutants
based on strength of phenotype) when the mutation is:
a.
Hypomorphic (Explain your reasoning for each example.)
b.
haploinsufficient
c.
antimorphic
5. The C. elegans lin-14 gene controls the timing of development in C.
elegans. LIN-14 protein is high early in development and gradually
decreases as development proceeds. lin-14 is defined by both
dominant and recessive mutant alleles. Animals that are
homozygous for recessive alleles develop precociously
(developmental events occur much earlier than normal) because LIN14 protein levels are lowered or eliminated, similar to the levels seen
later in development. Animals that contain dominant alleles are
retarded in development (developmental events occur much earlier
than normal) because LIN-14 levels are higher than they should be
late in development. You are given three lin-14 mutants.
a) Animals that are homozygous for mutation A develops
precociously and development is even more precocious when the
mutation is placed over a deficiency of the locus. What type of a
mutation is this?
b) Animals that are homozygous for mutation B develops
precociously and development is similar when the mutation is placed
over a deficiency of the locus. What type of a mutation is this?
c) Animals that are heterozygous for mutation C are retarded.
Development is less retarded when the C mutation is placed over a
deficiency and more retarded when the animals contain one C
mutant gene and two wild-type genes. What type of a mutation is
this?