Download Week 7 - Natural Selection and Genetic Variation for Allozymes

Week 7 - Natural Selection and Genetic Variation for Allozymes
Introduction
In today's laboratory exercise, we will explore the potential for natural selection to cause
evolutionary change, and we will study genetic differences among individuals. Both processes are
fundamental to Darwin's theory of evolution by natural selection. Under natural selection,
individuals that possess certain combinations of traits have a greater lifetime reproductive success
(usually measured as survival, mating success, or fecundity), than those that possess other
combinations of traits. To determine whether natural selection has occurred, one usually measures
features of the population before the selective event and then again after it. The strength of selection
is inferred from the difference in the characteristics of the population before versus after selection.
Natural selection is not sufficient to produce evolutionary change. Populations change only
when the differences among individuals are heritable, i.e. when they can be passed onto the next
generation. Differences among individuals can also arise from the environment that an individual
experiences. Biologists have learned that heritable, or genetic variation for important traits is
widespread in natural populations. The presence of this heritable variation is what causes the
population to change in response to a selective event.
In today's lab, we will first view a videotape that describes one case where evolution by
natural selection has been demonstrated. As you will see, the selective forces can vary from one
year to the next, so that features that are favored at one time may be disadvantageous later on. After
the film, we will examine the results of enzyme electrophoresis for populations of a Californian leaf
beetle (Chrysomela aeneicollis). The banding pattern on the gels is produced by one genetic locus
with multiple alleles. We will calculate allele frequencies in the population, based on the genotype
frequencies that we observe.
Activity 1: Watching the video 'What Darwin Never Saw.'
Introduction: This film describes the story of evolutionary change in Darwin's finches, which
inhabit the Galapagos Islands. Listen to the film carefully. After the film, discuss the following
questions with your lab partner(s) and then write your responses below. You will be asked to
explain responses to the rest of the class. You will also develop a question of your own to pose to
the class for discussion.
1a. What is the main feature that distinguishes the thirteen species of finches found on the Galapagos
Islands?
1b. What are the two fundamental elements of Darwin's theory of evolution by natural selection, as
described in the film?
1c. What effect did the drought of 1977 have on bird populations?
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1d. Which kind of seeds became much less abundant during the drought of 1977.
1e. What effect did the shift in seed abundance have on the finch populations?1f. Which seeds
became relatively more abundant during the subsequent El Niño event?
1g. How did the finch populations evolve 1) in response to the drought of 1977 and 2) in response to
the El Niño event?
1h. Your own question for discussion by the group.
Activity 2: Interpreting genetic variation at allozyme loci
Introduction
Mutations can cause changes in amino acid sequences. Individuals may therefore possess
different forms of enzymes. These different forms (allozymes) occur at the same frequency in a
population as the genes that code for them (unless there is a mutation that does not affect the
mobility of the enzyme).
One can use enzyme electrophoresis to study variation in allozymes. A tissue extract is
placed into a medium (such as a gel or a cellulose acetate sheet) and an electric field is placed over
the medium. After electrophoresis, the enzymes can be located by placing the gel into a staining
solution. This solution contains the compounds that the enzymes need to function, plus a coloring
agent. Over time, bands appear, which indicate the regions where the enzyme is located. If these
bands are in different positions, then the enzymes that produced them must have differed in amino
acid structure, indicating that genetic differences among individuals exist.
The enzymes that can be seen on a gel are called electromorphs. If a population has more
than one electromorph, the enzyme is said to be polymorphic. This indicates that at least two alleles
exist for the gene that codes for the enzyme. Many enzymes are polymorphic in natural populations
of organisms. We can use the variation in enzyme frequencies to study genetic variation in natural
populations.
These enzymes can therefore be used as markers of genetic variation. An important advantage of
these markers is that they are codominant (the phenotype of the heterozygote includes that of both
homozygotes). Because of their codominance, it is easy to score all genotypes in a natural
population (homozygotes and heterozygotes).
Examples of allozyme gels
We will look at two examples of the banding pattern produced on a gel. Samples from 10-12 beetles
were loaded at the bottom of the gel, and the enzymes moved upwards towards the top. We can
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name these enzymes in alphabetical order, with the fastest moving allele labeled 'F' (fast), and the
slowest moving allele labelled 'S' (slow). Generally we name the most common allele 'M' for
medium. Individuals with only one band are homozygous (e.g. MM), and those with multiple bands
are heterozygous (e.g. FM, FS). This is because the heterozygote will produce two different
enzymes that move at different rates through the starch medium when an electric current is passed
over them
Monomeric enzymes are those that function with only one subunit. When there are two or more
alleles in a population, a heterozygote will possess two bands, and a homozygote will possess a
single band. In this kind of codominance, the heterozygote shows bands only in the same locations
as the homozygotes. The picture shows a gel stained for the enzyme phosphoglucomutase. The
banding pattern for 10 beetles is shown. Four alleles are evident in the population (F, H, M, S).
Some of the genotypes are filled in the scoring sheet. Fill in the missing genotypes below.
1. _____, 2. _FM_, 3 _FS_, 4 _____, 5 _____, 6 _____, 7 _____, 8 _____, 9 _____, 10 _MS_
Dimeric enzymes function with two subunits. If there are two or more alleles in a population, then
the subunits may come together three different ways. The faster-moving form may combine with
itself or with the slower moving form. The slower moving form may also combine with itself or the
faster moving form. A heterozygote will therefore possess three bands, and in this kind of
codominance, you see bands in the same position as the homozygotes, but there is also an extra band
in the middle. The picture shows a gel stained for the enzyme phosphoglucose isomerase. The
banding pattern for 12 beetles is shown. Three alleles are evident in the population (F, M, S). Some
of the genotypes are filled in the scoring sheet. Fill in the missing genotypes below.
1. _FM_, 2. ___, 3 _MM_, 4 ____, 5 _MS_, 6 ____, 7 ___, 8 ___, 9 ____, 10 ___, 11 ___, 12 ___
Part 1: Interpreting electrophoretic variation at a monomeric enzyme. You will work in
groups of four
Each group will receive a picture of a gel that shows the enzyme banding pattern of about 30 insects.
You will also receive scoring sheets to identify the electromorph phenotypes on that gel. You need
to interpret these banding patterns. This will require the determination of the electromorph
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phenotype of each individual and counting the number of individuals that possess the genotype that
corresponds to each electromorph. Ask your instructor for help in your interpretation.
Name the electromorph phenotypes (= genotypes) in your sample
__________________________________
How many different alleles were present in the population? ___________
Part 2: Interpreting variation at a dimeric enzyme.
For the dimeric enzyme, you will receive a second picture of a gel that shows the enzyme banding
pattern of about 30 insects. You need to interpret these banding patterns as above for the monomeric
enzyme. Ask your instructor for help in your interpretation.
Name the electromorph phenotypes (= genotypes) in your sample
__________________________________
How many different alleles were present in the population? ___________
Part 3- Calculating observed genotype and allele frequencies for a dimeric enzyme
3a. Determine the number of individuals with each genotype and fill these values into column two of
Table 1.
3b. Divide the values in column 2 by the total number of individuals to obtain the genotypic
frequencies.
Table 1- Genotype numbers and frequencies.
Genotype
(number of individuals)
Genotype
frequency
(# individuals)
(total # in population)
Total
Calculate allele frequencies. First, you need to obtain the number of copies of each allele in the
population. Remember that each homozygous individual has two copies of each allele, while a
heterozygote has one copy of two different alleles.
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Column1
Col 2
Col 3
Col 4
Allele Number of Number of Number of Number of
homozygote heterozygot homozygote heterozygot
individuals e
alleles
e alleles
individuals
with this
with this
2 * Col 1
Col 2
allele
allele
Total
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Col 5
Total allele
number
Col
3 + Col 4
Col 6
Allele
frequency
Col 5
(2 * # individuals)