Part A: Yeast Genetics: Background

The Response of Yeast to Radiation

At the beginning of the 20th century, when physicists were trying to understand how matter and energy interacted, the classical theory of physics led to the conclusion that all warm objects should radiate all frequencies of electromagnetic radiation. In other words, everything should be shining with ultraviolet radiation. If nature behaved this way it would have been a catastrophe for life as we know it. Since the theory was absurdly wrong, scientists dubbed it the "ultraviolet catastrophe.". Because it led rather directly to the development of the quantum theory of light, however, it was anything but a catastrophe. Quantum theory gave us a way of understanding how molecules--including the important biological molecules such as DNA, chlorophyll, and visual pigments--interact with electromagnetic radiation. It gave us the concept of the photon as the elementary particle of light energy that interacts with atoms and molecules.
Today, the term ultraviolet catastrophe threatens to take on a more sinister meaning. If we have the potential to substantially increase the amount of solar ultraviolet radiation that reaches the surface of the earth, the consequences could be an ecological catastrophe. There are pessimists who will say that if there is a way to mess things up, surely we will do it. There are optimists who will say that even if we mess it up, we can turn around and fix it. We prefer to say that if we understand it, perhaps we can avoid messing it up to begin with.

 Ultraviolet radiation in our environment is as common as sunlight. It generates genetic diversity and kills cells. It gives us suntans and skin cancer. It appeals to our vanity and feeds our fears. In the classroom, ultraviolet radiation is a vital topic for the study of the global environment, health, genetics, evolution, chemistry, and physics (See Jagger 1985 and Part F: A Closer Look at...Ultraviolet Radiation in Our Environment).

 The amount of the damage that ultraviolet or ionizing radiations produce in growing cells is grossly disproportionate to the amount of energy that is absorbed. A lethal dose of radiation, for instance, will raise the temperature of a cell by only a small fraction of one degree. The reason for this incredible efficiency is that DNA is easily damaged by radiation and small injuries to the genetic material are amplified by replication and growth. In fact, the chemical DNA is even more radiation sensitive than one would guess from the sensitivity of the cell. Because of the environmental radiations that have existed on the earth for millions of years, cells have had to evolve machinery for repairing radiation damage to the DNA (Friedberg 1985).

 The mechanisms for repairing radiation damage, found in diverse organisms -- from yeast to humans -- are remarkably similar, suggesting that these repair processes evolved very early. The universality of these mechanisms makes yeast particularly useful for studying and demonstrating how most cells respond to radiation exposure (Haynes & Kunz 1981).

Ultraviolet and Ionizing Radiations

The physical mechanisms by which cells are damaged by ionizing radiations, such as x-rays and radiations from radioactive sources, is much different than the mechanisms by which ultraviolet radiation acts. Ionizing radiations, as their name implies, ionize molecules at random throughout the cell. Each ionization event -- actually a cluster of single-electron ionizations -- deposits, on the average, 100 electron volts of energy in a volume of space that is comparable in size to a molecule. This is enough energy to break many covalent bonds. These ionizations produce severe damage to the molecules. Ultraviolet, on the other hand, is absorbed by specific molecules and produces a limited number of specific types of chemical changes.

The biological effects of both types of radiations, however, are much more similar than their chemical effects. The sensitivity of a cell depends on the sensitivity of its most sensitive component -- often called a "target" by radiation biologists. In growing cells, the DNA is the most sensitive target molecule for both types of radiation. The biological sensitivity of a molecule depends on how readily it is damaged and how important it is to the cell.

Genetic Material is the Target for Both Types of Radiation

Although ionizing radiations are indiscriminate about which molecules they damage, the backbone chains of DNA are easily broken by ionization and there is enough energy in each ionization cluster to break both strands of the double helix. Such double-strand breaks are disastrous to the cell because they prevent the DNA molecule from replicating. Breaks in just one strand -- single-strand breaks -- are less serious.

 Ultraviolet, which does not break the DNA chain outright, is selectively absorbed by the aromatic rings of the purine and pyrimidine bases, so its energy, being more concentrated, is as damaging as ionizing radiation. One particularly unpleasant result is the formation of pyrimidine dimers. In this reaction, two adjacent pyrimidines in the same chain (T-T, C-C, or T-C) become covalently bonded together. These dimers disrupt the local structure of the DNA double helix and prevent normal DNA replication. They are not much better than a double-strand break, as far as the cell is concerned.

Repair of Radiation Damage

Radiation, however, is a natural component of the environment in which all living things have evolved. In fact, during the early periods of evolution, before there was as much oxygen (especially ozone) in the atmosphere, the amount of UV reaching the surface of the earth was probably much greater than it is today. Scientists believe that the oxygen in the atmosphere was mostly produced by photosynthetic plants, so these plants must have evolved before the ozone layer was present to screen out most of the UV. Nearly all organisms solved this problem by evolving mechanisms for reversing or repairing radiation damage. Such mechanisms are found in all organisms today and are essential for continued life.

There are several different types of repair systems. We can do a simple experiment to observe one of the most efficient repair systems in yeast, photoreactivation. When UV-damaged cells are exposed to sunlight that has the UV wavelengths filtered out, a specific enzyme in the cell uses the energy from the visible part of the solar spectrum to reverse the reaction that produces pyrimidine dimers. If the dimers are repaired before the DNA tries to replicate them, they have no effect on the cell. Another process, called excision repair, involves a sequence of events in which the damaged portion on one of the strands of the DNA double helix is removed by enzymes, and then the gap is accurately resynthesized, with the remaining strand acting as the template. Normal yeast cells have four mechanisms for DNA repair. Humans seem to have fewer types of repair mechanisms. In all organisms mutations often occur during less accurate repair processes, accounting for the mutagenic action of UV radiation.

 (See Part F: A Closer Look at Biological Consequences of Ultraviolet Exposure, and A Closer Look at...Repair of DNA).

Lethal Effects of Radiation

When a DNA molecule is damaged by radiation and the damage is not repaired before the DNA replicates, the cell is likely to die. When a cell cannot divide to form viable progeny, we say that it has suffered reproductive death. The cell may still be able to metabolize and grow, but it cannot divide. If the radiation dose is high enough, cells can be killed outright -- metabolic death -- but other metabolic functions are far more resistant than reproduction.

 The fate of an irradiated cell depends on the chance that its DNA is damaged and the chance that the damage is not repaired before the DNA is replicated. These are random chances, so the response of a population of irradiated cells can be described by the probability of rare events. For haploid cells, the probability of an irradiated cell dying is approximately proportional to the amount of radiation it absorbs. In the population, then, the fraction killed depends on the radiation dose. In diploid cells there is usually a threshold for radiation killing. Since each cell contains two copies of each kind of DNA, it is often necessary to damage both copies to kill the cell. There is an even larger threshold for killing multicellular organisms such as seeds, because it is necessary to kill more than one cell to kill the organism.

 With single cells, we can measure the number of individuals that are not killed more easily than the number killed. The survivors divide and form colonies, which we can easily count. With larger creatures we can count both survivors and non-survivors. With all organisms, however, we usually describe the results in terms of the percent of the population that survives a given dose. The results for a range of doses defines a survival curve. Figure 1 shows idealized survival curves for the three general cases, each plotted as percent survivors as a function of dose, and as log of percent survivors as a function of dose (semi-log plot):

Figure 1 : Idealized Survival Curves

A) Haploid cells normally have an exponential or nearly exponential survival curve. The slope of the exponential curve in a semi-log plot (when graphed on semi-logarithmic paper) is the probability per unit of dose of a cell being killed. The straight-line graph shows that the probability is approximately constant.
B) The survival curve for diploid cells usually has a shoulder at low doses. Small doses do not kill the cell, but produce sublethal damage. When enough damage is accumulated, the cell becomes more sensitive. At high doses the response becomes exponential. After the threshold is passed additional radiation has a constant probability of causing death.
C) Multicellular organisms have a broad threshold followed by a very sharp response. A large amount of cell death must occur before the whole organism is killed. The radiation dose has more effect on the time of death than on the likelihood of death.

Genetic Effects of Radiation

In cells, as well as in multicellular organisms, the individuals that survive radiation exposure are not necessarily unaffected. There are many long-term effects in multicellular organisms, the most significant being increased risk of cancer. There are other, more poorly defined effects that can be measured by their reduction of life expectancy. In single-celled organisms such as yeast, other fungi, bacteria, and algae, mutations are an important sublethal effect. Radiation also produces sublethal chromosomal changes and stimulates genetic recombination.

 In complex organisms, where the cells involved in heredity -- germ line cells -- are distinct from the cells that form the rest of the organism -- somatic cells -- the consequences of genetic changes depend largely on the type of cells damaged. Germ-line-cell mutations produce heritable changes that may be passed along to progeny, whereas somatic-cell mutations may have no effect at all, or may cause diseases such as cancer, or activate dormant viruses.

 In single-celled organisms, such as yeast, where this differentiation does not occur, all types of genetic changes can be studied more directly at the cellular and molecular level. Most nonlethal mutations are recessive, and are most easily studied in haploid strains, where they are expressed directly. Dominant mutations, on the other hand, can be studied in diploids. The stable haploid phase in the life cycle of yeast makes them especially convenient for mutation studies. Although most higher plants and animals have only diploid somatic cells, the wasps and bees (hymenoptera) are an important exception, and have been used for mutation studies. While females of this order are diploid, the males are normally haploid. Males develop by parthenogenesis from unfertilized eggs while females develop from fertilized eggs.

Artificial Sources of Ultraviolet Radiation

Artificial lights that emit ultraviolet radiation can be used for biological experiments. The two general types are incandescent lights and fluorescent lights. With appropriate precautions, you can use artificial light as a UV source in your experiments.

Incandescent Lights
When the tungsten wire filament of an ordinary light bulb is heated by an electric current it gets hot enough to emit light, and some of that light will be ultraviolet. At higher temperatures, more of the light is emitted as ultraviolet. A type of bulb known as a quartz-halogen lamp has a tungsten filament inside a quartz tube filled with an inert halogen gas. Quartz-halogen lamps can be heated to higher temperatures than ordinary light bulbs, and give off an intense light containing a considerable amount of UV-A and UV-B, and even a little UV-C. Such light bulbs are commonly used for automobile headlights, slide and overhead projectors, and outdoor security lights. Figure 2 shows the energy spectrum of a 300 watt quartz-halogen lamp that is available in hardware and discount stores for less than $20.
Normally, quartz-halogen lights are operated in a glass enclosure, which absorbs the damaging UV photons. If one removes their protective glass cover, however, they become a source of artificial sunlight. The spectrum of light they emit is quite similar to that emitted by the sun after it has been filtered through the atmospheric ozone. With its glass cover open, the lamp whose spectrum is illustrated in Figure 2 can be used as a substitute for sunlight. With the cover closed, it can be used as a source of visible light for photoreactivation.
Obviously when the protective glass cover is removed, a quartz-halogen lamp is hazardous, since it emits as much damaging UV energy as bright sunlight, so those working around it must protect their eyes and skin from the direct radiation.

 Fluorescent Lights
Ordinary fluorescent tubes operate when a high voltage electric current passes through a tube filled with mercury vapor. Most of the radiation given out by the mercury atoms has a photon energy of 4.8 eV, which is in the UV-C range (wavelength equal to 254 nm). Ordinary "visible light" fluorescent tubes are enclosed in glass that is coated on the inside with a fluorescent material that absorbs the high energy UV photons and re-emits the energy as visible light photons. Very little of the UV escapes.
When different types of fluorescent coating materials are used, the tubes can be engineered to emit different ranges of photon energies. The common "black lights" used to display fluorescent posters emit their energy in the UV-A region. Lamps that emit UV-B are commonly used in research for observing fluorescent dyes in DNA and proteins. These UV-B lamps, which are more intense sources of UV-B than the quartz-halogen lamp, can also be used to kill the sensitive yeast strain (G948-1C).
A particularly powerful lamp that looks like a fluorescent tube is called a germicidal lamp. This is a "fluorescent" tube without any fluorescent coating and a tube that will transmit the 4.8 eV photons of the mercury vapor. A germicidal lamp is the only common UV source that is energetic enough to overpower the normal yeast repair systems. It is commonly used in research to generate mutants. Figure 3 shows the energy spectra of each of these three types of lamps.

Explore UV sources in the computer program UVRISK, (See Part F: A Closer Look at...Modeling the Effects of Ultraviolet Radiation and A Closer Look At... Light and Energy.

Where you can buy UV lamps
The quartz-halogen lamp that we have used is Model 3030 Quartz Halogen Lamp Fixture manufactured by Lights of America, Walnut, CA 91789. It is distributed through K-MART stores and some hardware stores. It sells for $10 to $20 and must be wired to a power cord. Mount it on a secure stand at a distance at least 8 inches above the Petri plate, to keep the heat at an acceptable level. At this distance an exposure of 3 minutes will give a survival fraction of 0.1 with the sensitive strain G948-1C.
Fluorescent UV lamps and germicidal lamps are sold by many vendors. One source that carries all three of the lamps whose spectra are illustrated in Figure 3 is Cole Parmer Instrument Co. (800-323-4340). They sell 15 watt tubes under the following catalog numbers: G09815-55 (UV-A), G09815-63 (UV-B), and G09815-59 (UV-C). A safe enclosure for using these lamps is described in this volume.


CAUTION! UV-B and UV-C lamps are DANGEROUS!

 Germicidal lamps, and to a lesser extent, UV-B lamps, are dangerous. Brief exposures can produce serious burns to skin and eyes. Always use them in a safe enclosure that prevents accidental exposure. A detailed plan for such an enclosure is described in this handbook.

Figure 2

 (See Part F: A Closer Look at...Ultraviolet Radiation in Our Environment).

Figure 3: Energy spectra of a quartz-halogen yard light with the glass cover open and with the cover closed.

 FIGURE 3: Energy spectra of germicidal and florescent UV lamps.

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Last updated Wednesday, 04-Dec-2002 20:46:18 UTC