DNA Damage- The Impact of UV Light
AUSTRALIAN CURRICULUM ALIGNMENT:
- Variations in the genotype of offspring arise as a result of the processes of meiosis and fertilisation, as well as a result of mutations (ACSBL084)
- Mutations in genes and chromosomes can result from errors in DNA replication or cell division, or from damage by physical or chemical factors in the environment (ACSBL082)
- Mutation is the ultimate source of genetic variation as it introduces new alleles into a population (ACSBL092)
We classify the broad spectrum of electromagnetic radiation from the sun into segments according to the effects we experience. For example, the warm sensation of sunshine on our skin is caused by invisible infrared radiation with wavelengths ranging from 700nm to 1,000,000nm (1mm). Visible light is comprised of wavelengths between 400nm (violet) and 700nm (red). Radiation with wavelengths shorter than 400nm but longer than 10nm is classified as Ultraviolet (UV) radiation. Radiation with wavelengths shorter than 10nm are classified as X-Rays. Some exposure to UV radiation is necessary for humans to produce vitamin D, but a careful balance is required because X-Rays and UV radiation are destructive to many biological molecules, including DNA. Fortunately, the earth’s atmosphere acts as a protective screen and filters out almost all the sun’s radiation with wavelengths shorter than 290nm. Nevertheless, the narrow UV band from 290nm to 400nm that can penetrate the atmosphere and reach the surface of the earth is capable of causing photochemical damage to DNA that can lead to skin cancer, so it is important to avoid over-exposure. As a defence against too much UV exposure, most organisms that are subject to the sun’s rays have evolved to incorporate some level of DNA repair in their cell mechanisms. This confers a limited amount of inherent UV resistance.
In this practical, students will expose the mutant strain of yeast, Saccharomyces cerevisiae, to UV. This strain does not incorporate all the genes necessary for effective repair of photochemically damaged DNA. As a result the exposure to sunlight kills this strain of yeast more quickly than wild type Saccharomyces cerevisiae. This is an excellent opportunity for students to observe how UV radiation can be destructive for many biological molecules.
PREPARATION- LAB TECHNICIAN
Prepare YED plates:
- Melt the contents of the Melt and Pour bottles in a beaker of gently boiling water.
- Aseptically pour into sterile petri dishes. Allow 25mL per plate, which means you can expect to pour 4 plates per 100mL bottle of Melt and Pour YED agar.
- After pouring, allow the YED plates to cool on the bench. When set (about 30 minutes), turn upside down and store for up to two weeks under refrigeration.
- Take the plates out of the fridge about an hour before use so they can warm up to room temperature. If there is any condensation on the underside of the lid, remove it by carefully flicking the water off.
- In order to ensure you are working with vigorous yeast cells in the exponential growth phase, it is necessary to prepare a starter plate. The timing will depend on the incubation temperature you plan to use.
- Prepare 48 hours before exposure if incubating at 30°C, or 4 days before exposure if incubating at room temperature (24±4°C). In practice, the timing of the experiment will determine how you proceed. For example, to be able to do the student experiment on a Wednesday, you should prepare your starter plate on the Monday and incubate at 30°C for 48 hours.
- If you will be doing the student experiment on a Monday, you should prepare your starter plate on the previous Thursday and incubate for 4 days at room temperature. Work out a temperature/time regime that suits your equipment and schedule.
- The main thing is to keep the starter plate in darkness and use it at the appointed time. If you extend the incubation time (longer than 48 hours at 30°C or longer than 4 days at room temperature), the exponential growth phase will have passed and the cells will not be ideal for the student experiment.
- Prepare your starter plate: Use a sterile inoculation loop to aseptically transfer a UV sensitive yeast sample from the slope supplied to a fresh YED plate (free of condensation). Follow the technique required to generate single colonies.
- Repeat above step to create a ‘control’ plate using Saccharomyces cerevisiae.
- One starter plate of each yeast strain will be enough to supply yeast samples for several student groups, but it is prudent to make a number of starter plates (up to one per student group) in case one or more fail to grow, and to enable students to work simultaneously without having to wait for a group to finish and pass the starter plate on.
METHOD-STUDENT ACTIVITYInoculation of Exposure Plates:
- Use a sterile transfer pipette to place 1mL of sterile water into a sterile tube.
- Use a sterile inoculation loop to carefully scrape a single colony of yeast from the starter plate.
- Select a large colony (>4mm in diameter), or if the colonies are small, scrape up 2, or even 3 onto the loop.
- Place the loop in the water in the sterile tube and spin/swirl it to transfer the yeast into the water.
- Visually check that the cell mass has transferred from the loop to the water.
- Immediately use a 1mL sterile pipette to pump the liquid in order to distribute and suspend the yeast cells in the water. Avoid entraining air bubbles or splashing the liquid up the sides of the tube. When finished, hold the tube up to the light to check that there are no visible lumps or particles in the water.
- Dip a sterile swab into the yeast suspension and as you withdraw it, press it against the sides of the tube to squeeze out excess water. It should come out moist but not dripping.
- Using aseptic technique, “paint” the surface of a YED plate in three directions to inoculate for a lawn culture.
- Cover the plate to shield it from light and allow it to rest (right way up) for a period of at least 15 minutes and up to one hour. This allows the moisture from the swab to be absorbed by the agar.
- Prepare as many inoculated plates as required.
Exposure to Sunlight:
After the post-inoculation resting period, expose the inoculated plate to direct sunlight to observe the degrading effect of UV light. Immediately after exposure, incubate the plates in darkness for 48 hours at 30°C or 4 days at room temperature. For best results, follow these guidelines:
- Keep the plate shielded from light until the last moment.
- Use adhesive tape to attach the lid of the petri dish to the base, but do not allow the tape to extend on to the surface of the lid where it will absorb UV light and shield the yeast from exposure. Write identifying notes on the base of the plate.
- Orient the plate so the lid is pointing directly at the sun. Aim to minimise the size of the shadow. If the sun’s rays strike the lid at a glancing angle, most of the UV light will be reflected and the effectiveness of the exposure will be reduced.
- Schedule the activity at a time of year when you can be sure of bright sunny conditions. Late Spring or Summer or early Autumn are best.
OBSERVATION AND RESULTS
Effectiveness of Sun Blocks
- Prepare a series of plates inoculated with UV-Sensitive Yeast.
- Obtain a number of commercial sunscreens (e.g. with different SPF values, or different brands of the same SPF value) and/or other materials (e.g. oils such as olive, coconut, canola, or materials such as cellophane and different fabrics, or different sunglasses – challenge students to come up with ideas to test).
- Apply the test materials to one half of the lid of the plates and expose the entire plate to UV light for a set period.
- Carry out a preliminary evaluation to determine a suitable exposure time (that will kill a significant proportion of the unprotected yeast cells). Include a control that completely masks half a plate (e.g. opaque cardboard) to ensure no light reaches the UV-sensitive yeast.
Obtaining Numerical Results
- Prepare a suspension of UV-sensitive yeast cells by transferring a colony from a starter plate to 10mL (accurately measured) of sterile water.
- Use a micropipette to produce five more vials in a serial dilution ranging from 10-1 to 10-5. Use a micropipette to transfer 150uL of each suspension to a YED plate (total of 6 plates) and spread with a sterile spreader (one per plate).
- Incubate in the dark and identify the dilution for which the plate has around 30 to 120 colonies.
- This is enough colonies to be easily counted without being too many to count.
- Repeat the procedure but this time produce 6 plates at the dilution level identified as having around 30 to 120 colonies.
- Carry out an evaluation to determine how long you need to expose the plates to kill all the cells.
TEACHER TIPUV sensitive yeast is degraded by exposure to UV light. Although indoor lighting and indirect sunlight contains very low, if any, UV radiation, we recommend ensuring minimal exposure by:
- Drawing down blinds if working near windows
- Switching off unnecessary lights when working under interior lighting
- Covering the UV sensitive yeast culture with opaque foil whenever possible.
- 55 mins
- UV Sensitive Yeast Live Slope
- Wild Yeast, Live Slope
- Sterile inoculation Loop
- Sterile Petri Dishes
- Sterile Pipette
- Culture Tubes
- YED Agar (Melt and Pour)
- Bunsen Burner
- Adhesive Tape
- Permanent Marker
- Wear appropriate personal protective equipment (PPE).
- Know and follow all regulatory guidelines for the disposal of laboratory wastes.
- Wash your hands thoroughly after the activity.
- Protect yourself from exposure to UV and ensure you know what wavelengths of UV your artificial lights emit.