What repair proteins are expressed in C-ferns after exposure to varying amounts of UV radiation?
Forest Hills HS, Queens
Summer Research Program for Science Teachers
Summer 2006
Materials:
1) UV Lamp – one that has UVA and UVB rays.
2) Supplies for culturing C-ferns can be obtained from Carolina Biological.
3) Protein electrophoresis units and supplies can be obtained from BioRad.
Precast gels -12% or 15%.
Tris – glycine SDS Buffer
Laemmli’s Sample Buffer
Kaleidescope Precision Standard
Silver Staining Kit or Coomassie Blue Stain
(Also needed are: micropipets, microtubes, and microcentrifuge.)
4) Vernier UVA and UVB probes.
A Computer to download probe software.
5) A stereoscope (dissecting microscope) to observe the c-ferns.
6) UV protection goggles.
7) An enclosed box for UV radiation.
8) Spectrophotometer – optional for extension work.
Background:
Depletion of the ozone layer and subsequent increased exposure to UV radiation is a very relevant environmental issue today. How do plants respond to this increase in UV radiation? What DNA repair mechanisms are in place in most plants? There is much literature available on the UV repair mechanisms in angiosperms and higher plants, but what about lower plants, like c-ferns? Students may want to do some research on DNA repair mechanisms in Arabidopsis thaliana as a pre-lab assignment. This is a model plant organism for which there is a wealth of online information. Students will find several DNA repair proteins, the most prominent one being the enzyme photolyase. Have students find the molecular weight of photolyase in Arabidopsis. This will give them a ball park range for determining of photolyase is actually present (and in increased concentrations) in their treated specimens when they do the protein electrophoresis. Have students find the molecular weights of other repair proteins they find in the literature.
Students will also need to study the life cycle of c-ferns if they have never worked with this organism before. Ferns have a prominent gametophyte and sporophyte stage. (Some students may want to expose the gametophyte to radiation while others expose the sporophyte to UV radiation to see if there are different proteins expressed in these different stages.)
Procedure:
1) Grow the C-ferns
Make the c-fern media using sterile technique. Media can be purchased from Carolina Biological in the powder form or already made, in bottles. The powder is less expensive, with one packet making a liter of media. Instructions for making the media are included with the packet. Purchase wild type spores (one vial is usually enough to inoculate 30 small Petri dishes) and hydrate when the plates have been filled with media. Inoculate the plates. (Some UV resistant, mutant spores are also available from Carolina. Some students might want to perform this experiment using these mutant spores and comparing their electrophoresis results with the wild type results).
When lighting and temperature conditions are optimal the gametophytes should be ready to be exposed to radiation in a week to 1 ˝ weeks. The gametophyte (hermaphrodite) will have a heart-shaped appearance at this point. The cultures can be observed using a dissecting microscope.
2) Exposing the Gametophytes
Have different groups of students vary the UV exposure time (from 5 minutes to an hour). All exposure should be done inside an enclosed box. All plastic covers must be removed from the culture plates. Line the inside of the box with aluminum foil. Wipe the aluminum foil with ethanol before opening the culture plates to decrease contamination. Insert the Vernier probes to generate a graph of the strength of the rays generated on the computer. Some of the groups may want to add regular white light along with the UV light into the UV chamber as a control. This will eliminate the possibility of lack of white light being a cause of plant deterioration. If this is done, use a blue light filter on the white light, since blue light waves will activate the photo-repair mechanism.
3) Collecting Specimens for Protein Electrophoresis
Some specimens should be collected immediately after UV exposure for electrophoresis. A second batch of specimens should be collected 24 hours after exposure for electrophoresis. Collect about 20 – 30 gametophytes. Weigh the gametophytes. Be certain that both the first collection and the second collection of gametophytes are exactly the same weight. Discrepancies in weight will invalidate any comparisons you want to make with your electrophoresis results. Add 200ml -250ml of Laemmli’s sample buffer to the microtubes. Homogenize the specimens in the buffer until the plants form a pulp. Spin in a microcentrifuge and pipet off the supernatant. If there is no liquid supernatant after homogenizing, add some more Laemmli’s buffer. (Add this same amount of extra Laemmli’s to your other samples to keep the concentration constant.) This supernatant will be used for the electrophoresis.
4) Protein Electrophoresis
Set up the gel boxes according to the instructions of the manufacturer. Place 15 ml of each sample into a different well. One well must have 10 ml of the standard (Kaleidescope). Run the gel at 200 volts for about 30 minutes.
5) Staining
A general stain may be used, like Coomassie Blue or a more specialized stain like the Silver Stain, obtained from Bio Rad. The Silver Stain is more sensitive than the Coomassie Blue. If you suspect that you have a small amount of protein of interest in your sample it is better to use the Silver Stain. Follow the directions that come with the Bio Rad Silver Stain Kit.
6) Using log paper, generate a “best fit line” from the molecular weight standards. In the standard lane, measure the distance of each band from the bottom of the well. Once the standard bands have been plotted the bands of the sample wells can be compared to it. You can now make predictions about the molecular weights in your samples.
7) Students can compare the banding patterns of the repair proteins they find on their gels, contrasting the control and UV exposed samples.
What UV repair proteins were expressed?
What are the relative concentrations of these proteins in the different samples?
Are the repair proteins expressed immediately after exposure to UV or is there a lag time after exposure?
8) There are several follow-up or extension procedures that can be done following the above experiment.
An extension of the lab may be to do spectrophotometric analysis of the different samples to determine if there is a loss of chlorophyll in the UV treated specimens.
Another extension would be to continue the growth of the plants and observe if there is complete recovery of the plants over time. If not, what may be some factors contributing to the lack of recovery?
Standards:
Teaching Standard A
Teachers of science plan an inquiry-based program for their students. In doing this, teachers:
(1) develop a framework of yearlong and short-term goals for students.
(2) select science content and adapt and design curricula to meet the interests, knowledge, understanding, abilities, and experiences of students.
(3) select teaching and assessment strategies that support the development of student understanding and nurture a community of science learners.
(4) work together as colleagues within and across disciplines and grade levels.
Content Standard A
As a result of activities in grades 9-12, all students should develop :
(1) abilities necessary to do scientific inquiry
(2) understandings about scientific inquiry
Content Standard C
As a result of their activities in grades 9-12, all students should develop understanding of
(1) the cell
(2) molecular basis of heredity
(3) interdependence of organisms
(4) Matter, energy, and organization in living systems
(5) behavior of organisms
Content Standard E
As a result of activities in grades 9-12, all students should develop:
(1) abilities of technological design
(2) understandings about science and technology