Download General Introduction

Mendel’s Wrinkled Peas
General Introduction
You are already aware that the information contained in a gene can affect properties of an organism.
Phenotype is the term used to describe these properties. Keep in mind that a phenotype can be something
very easily observable (such as human height) or more subtle, requiring instruments to do the observing (such
as the amount of an enzyme produced inside the cells). Sometimes a single phenotype is produced by the
combined effects of more than one gene. Sometimes a single gene affects more than one phenotypic
characteristics. So, the picture can be quite complicated.
Mendel found that pea plants with alternative forms of a single gene can produce pea seeds that differ
in their shapes: smooth or wrinkled. As you will see in the work for today, this characteristic is only one of
several that are alterable by a single gene. Along the way, you will recognize that the pathway between gene
and final outcome(s) consists of multiple steps. This applies to any gene, not just the one you will be studying.
Keep Figure 1 before you as you carry out the work today; it shows the hypothetical multiple steps that are
influenced by the gene.
Specific Introduction
The “smooth-wrinkled” gene will be symbolized by either a W (standing for the dominant allele that
produces a smooth exterior) or a w (for the alternative allele producing wrinkling if the seed is allowed to
become dry). This gene produces an enzyme involved in the production of starch in the developing seed. You
will remember that starch is a polysaccharide composed of many glucose molecules that become covalently
linked. More specifically, the W allele produces an enzyme that causes “early embryonic” branching of the
starch molecules. The same enzyme also influences the expression of other enzymes (coded by other genes)
that are also involved with starch production. The other allele, w, makes an alternative form of enzyme that is
less efficient in doing early embryonic branching of starch. The result is that there is less starch made in a seed
with only the w allele present. If there is less starch made, this means that a ww individual contains more free
glucose; it wasn’t converted to starch.
What does this have to do with the shape of a seed? While the peas are still in their pods (that is,
before they are released and become dry), they can accumulate water from nearby tissues, by osmosis. You
know that osmosis is influenced by the amount of dissolved small molecules and ions inside of a cell; the more
there are in the cytoplasm, the greater the osmotic potential, the more water will enter the cell. Peas with
genotype ww have not converted much glucose (in the form of glocuse-1-phosphate) into insoluble starch, so
they have greater osmotic potential and swell up more than peas with the W_ genotype. These peas will tend
to lose more water during subsequent drying (since they had more to lose), and their surfaces will become
quite wrinkled. (The outer coating of seeds is not very elastic.) Peas that did not gain as much water in their
early stages of development will not grow as large a surface and will not shrink as much during drying; they will
keep much of their original round shape.
To study the multiple effects of the gene, you will do several things today. (1) You will extract the
contents of peas to test for the presence of a specific enzyme, call starch-branching enzyme I (SBE1). This
enzyme is responsible for adding individual molecules of glucose to make long branching chains of starch. The
W allele causes early production of SBE1, and the finished pea should have little of this enzyme left. Plants
with the w allele also make a form starch-branching enzyme, but it appears quite late in development, after
much of the opportunity to make starch has been lost. These peas should have more of this type of enzyme
still present, since it was made later. (2) Whether starch granules were made early or late determines the
shape of these stored granules. You will use a compound microscope to see the granules, compared the
shapes, and see if there is a correlation between shape and genotype. (3) Finally, you will test for the osmotic
potential of the smooth and wrinkled peas, by measuring amount (by weight) of water uptake as dried peas
are placed in a watery environment.
Genetics
1
Lab
Figure 1: The relationship between W and w alleles and their influence on the phenotype.
w
DNA
W
transcription
RNA
translation
Polypeptide
Starch branching enzyme
Glucose-1-Phosphate
Efficient form of
the enzyme in
early
development
High starch
output,
Low sugar
content
Starch branching enzyme
Glucose-1-Phosphate
Inefficient
form of the
enzyme in
early
development
Low starch
output,
High sugar
content
“compound”
starch grains
“simple”
starch grains
Normal
osmotic
potential
Altered
metabolism,
late in seed
development
Normal
water
content
Other starch
producing
enzymes
stimulated
Remains
plump
during
dehydration
Increases
osmotic
potential
Water
accumulates
Shrivels
easily during
dehydration
Increased
cell size
WRINKLED
ROUND
Figure 2. How
starch branching
enzymes bring
glucose together.
Genetics
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Lab
Procedures Work in groups of two or three, as instructed.
I. Extraction
1. Obtain 4 presoaked peas of each type. Label 3 test tubes with the initials of the pea type and add your
own identifying mark (e.g., your initials).
2. Place any one of the varieties of peas in the mortar and add 10 ml distilled water. Use the pestle to
thoroughly grind these seeds until no larger pieces remain. Usually, this requires that you push hard
while twisting the pestle against the mortar.
3. Pour only the liquid into the appropriately marked test tube. If some of the granular mash gets into
the test tube, it will not be a problem.
4. VERY IMPORTANT! Thoroughly wash the mortar and pestle with several rinses of tap water and a final
rinse of distilled water, to prevent cross-contamination between samples.
5. Repeat step 2-4 for the other two pea varieties. (This step may get modified. Pay attention during prelab lecture.)
II. Analyses
1. Obtain 3 Pasteur pipettes and rubber bulbs. Label each pipette with the initials of one of the pea
varieties. Draw up some liquid from each strain’s extract and add a drop to each of two clean
microscope slides. Label the slides. Add a drop of I-KI (iodine and potassium iodide, a specific stain for
starch and other polysaccharides) on just one of the slides, and cover the drops with coverslips.
Repeat with the other varieties; you should now have 6 slides. Set them aside for the moment.
I-KI
Pea
extract
2. When all research groups have completed steps 1-5 of extraction, place your 3 test tubes with those of
other groups into a clinical centrifuge. Spin the centrifuge at about 2500 rpm, for 10 minutes.
3. While waiting for the centrifugation to be done, go back to the slides. Using a compound microscope
at 100X magnification, look at the granules on the unstained slide. Then, go over to 400X
magnification for a better look. Compare the stained and unstained slides, to confirm that the
granules are actually made of starch. (However, you will no be able to reliably discern differences
among the three pea types based on degree of staining.) Looking at the unstained slides, try to
determine whether the three pea types have differences in size of individual granules, in general shape
(Smooth? Jagged?) of granules, and in cohesiveness of granules with each other, Make good drawings
of what you see.
4. Obtain a petri plate with agar that contains glucose-1-phosphate. This is the form of glucose that a
starch-building enzyme can use to make the long chains of glucose that are called starch. Turn it
upside down and use a marker to divide the bottom of the agar plate into 4 equal sections. Label the
quadrants C (control), EA (Early Alaska), LM (Little Marvel) and TL (Thomas Laxton).
5. After the extracts have been centrifuged, use 3 clean Pasteur pipettes to transfer one drop from each
suspension to its corresponding position on the agar plate. (Be sure to handle the centrifuge tubes
gently to avoid re-suspending the material that had been spun down.) Use a 4th pipette to transfer one
drop of distilled water to the quadrant labeled C.
6. Set the agar plate aside for about 30 minutes (using care to keep the drops from spreading out of their
quadrants). After 30 minutes, place a drop or two of I-KI onto each of the four sample drops you had
placed earlier, to test for the presence of starch (and therefore, for the presence of starch-building
enzyme in the extracts). Watch for any color change.
Genetics
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Lab
7. Take a sample of 5 dried peas form each strain. Then, weigh each group of seeds. Record the weights
below. Repeat, with a sample of 5 peas from each strain that had been soaked in water overnight. Dry
the peas first, using paper towels. Record these results. Calculate the percent increase in weight for
each variety of pea. (Note: Of course, you are not weighing the same 5 seeds before and after
treatment, but you can pretty safely assume that each group was chosen randomly and that any
random sample of 5 dry and 5 wet seeds reflects typical weight differences. Ideally, we would use the
same seeds before and after treatment.)
Journal of Results
1. Shape of dry peas:
Pea Shape
(Round or Wrinkled)
Pea Color
(Yellow or Green)
Early Alaska
Thomas Laxton
Little Marvel
2. Appearance of starch grains (make drawings):
Early Alaska
Thomas Laxton
Little Marvel
3. Agar Plate. Indicate whether each pea type was positive for starch, and if so, how strongly positive.
EA:
TL:
LM:
Control:
4. Weights of seeds:
Total dry weight
Total wet weight
Early Alaska
Thomas Laxton
Little Marvel
Genetics
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Lab