Mendel was definitely on to something, but to progress further, a better biological model than pea plants, or any other type of plant for that matter, would be needed. Enter the common fruit fly.
Thomas Hunt Morgan (1866–1945) was a prominent American biologist on the faculty of Columbia University, which was considered by many to have the premier biology department in the United States at the time. He was among the first to recognize that fruit flies are an ideal model for performing heredity research in animals.6
In many respects Morgan was the antithesis of his future protégé, Muller. They had very different personalities, perhaps owing to their notably different backgrounds. While Muller was self-conscious about his first generation immigrant status, Morgan had a pedigree that was none too shabby. His father’s side of the family was originally of Welsh descent. They had settled in Kentucky, opened a store and trading route, and soon became one of the wealthiest families in the state. Not only that, virtually all the men in the family had been war heroes in either the Revolutionary War or the Civil War (fighting on the Confederate side). His uncle, John Hunt Morgan, in particular, was nearly worshipped as a fallen hero of the Confederacy in his hometown of Lexington. Morgan’s mother also had blue bloodlines. She came from the aristocratic Howard family of Maryland. The Howards had long been involved in Maryland politics, with Morgan’s grandfather once serving as governor. And his great grandfather was Francis Scott Key, the famed lyricist of the Star Spangled Banner. None of this pomp phased Morgan. He never talked much about his family heritage, preferring instead to discuss biology, his true passion.
Morgan’s famous “Fly Room” was a laboratory where he conducted his genetic research on fruit flies.7 He had previously experimented with dozens of different types of animal species as models for inheritance studies, but each had its drawbacks. Ultimately, he settled on the fruit fly as the most practical genetics research tool, for reasons that will soon become apparent.
Morgan was a questioner by nature, but his doctoral training had made him an even greater skeptic. In 1886, he was among the first graduate students enrolled at the then ten-year-old Johns Hopkins University in Baltimore, a new type of graduate university founded exclusively upon a research premise, which was a novel educational concept at the time. There were no formal lecture classes, only laboratories. As a learning exercise, before starting any original research on their own, students were required to select some important published research in their field and repeat the experiments themselves to either verify or refute the findings.8 All scientific dogma was questioned, and nothing accepted without replication. Given this mindset, it is not surprising that Morgan was skeptical that Mendel’s findings in pea plants were relevant to animals. He decided to repeat Mendel’s inheritance experiments, but using his fruit flies instead of pea plants as the model organism. He didn’t expect to find much.
Morgan soon proved himself wrong about Mendel’s work. Using his fruit fly model, he discovered, to his own astonishment, that the traits of fruit flies transmit from generation to generation in a pattern that exactly corresponds to Mendel’s principles. That is, the pattern of inheritance that Mendel saw for tall versus short pea plants was exactly the same as Morgan saw for long versus short wings. And all of the second-generation hybridizations and back-crosses with the flies produced offspring in the same ratios as found for Mendel’s peas. Morgan then analyzed other traits in the flies and they behaved similarly. At this point, Morgan, too, became an unabashed disciple of what had become known as Mendelianism.
Morgan did more, however, than just show that Mendel’s work in plants could be repeated in an animal model. He took Mendelianism to the next level. He was able to convincingly demonstrate that the pattern of gene transmission from one generation to the next corresponded to the pattern of chromosome transmission, causing him to make the conclusion that genes were not just a scientific conceptualization; they were actual physical entities that resided on chromosomes, the threadlike structures found in the nucleus of cells. This insight proved to be absolutely correct, and would earn him the Nobel Prize in Physiology or Medicine in 1933. Genes were real things that could potentially be isolated, manipulated, and studied. They were also things that could be damaged.
FIGURE 10.1. IDENTICAL GENETIC TRANSMISSION IN PEAS AND FRUIT FLIES. Thomas Hunt Morgan found the exact same genetic inheritance patterns in fruit flies that Gregor Mendel had previously reported in peas, suggesting that the fundamental mechanisms of genetics were universal for all species, whether plant or animal. When Mendel crossed yellow peas with green peas, the first generation (F1) of peas were all yellow, but the second generation (F2) were 3/4 yellow and 1/4 green. Morgan found the same ratios for hybrid crosses of fruit flies with different eye colors. Crossing red-eyed flies with white-eyed flies resulted in all red-eyed progeny in the first generation, but 3/4 red-eyed and 1/4 white-eyed progeny in the second generation. These findings ultimately resulted in the fruit fly becoming the standard animal model for studying the genetic effects of radiation.
After completing his undergraduate studies, Muller stayed on at Columbia to obtain his PhD, and Morgan became his doctoral mentor. The two men even ended up coauthoring a book entitled The Mechanism of Mendelian Heredity.9 It was considered by many to be the seminal textbook on Mendelian genetics, articulating Mendel’s principles better than even Mendel had. Morgan even sold Muller on the idea that fruit flies were the best tool for genetic research. Nevertheless, the two men would never become friends. Muller felt that his mentor, Morgan, did not give him enough recognition for the brilliance of his ideas. Morgan, who was data driven, thought ideas were cheap to come by and preferred to heap his praise on those students who had actually toiled to produce the data needed to test hypotheses. Ironically, Morgan, for all his aristocratic roots, identified more closely with the workers than the intelligentsia.10
Tension between Morgan and Muller persisted, and eventually the two men went their separate ways. Still, both remained devoted to fruit fly research for the rest of their lives. What was it about fruit flies that made them so special?
If you want a pet, buy a dog. If you want meat, raise pigs. If you want to do genetic research, however, get yourself some fruit flies. For fruit flies, their virtues are in their numbers. They have very short generation times (10 days), produce large numbers of offspring per generation (about 500), and are very easy to care for and manipulate. Give them a piece of banana to eat and a small jar for a home, and they do quite nicely. It is also easy to distinguish the males from the females (by a dot on their abdomen). Best of all, unlike dogs and pigs, they don’t bite.
Fruit flies enabled genetic research that could not be directly performed in higher animals or humans. Consider this: humans have generation times that average about 20 years, so the number of generations of humans since 15 AD (i.e., 2,000 years ago) has been about 100. In contrast, an equal number of generations of fruit flies can be recapitulated in less than three years. And the offspring from a single pair, magnified over those 100 generations, would produce many times more descendants than there are people on Earth. In short, if a lot of numbers in a brief period of time are required, fruit flies can deliver.11
This is particularly important if your interest is in inheritable mutations. These altered traits are sometimes benign or even advantageous, but usually are harmful to the offspring. Mutations occur naturally and randomly in all populations of plants and animals, but they are very rare. It took two years for Morgan to find his first mutant, a white-eyed fly, among the hundreds of thousands of flies that he reared in his lab.12 (The eyes are normally red.) So, if you want to find mutants, you have to look very hard. This is why fruit flies are useful for studying mutations. Since there are so many flies available to examine, if you have the patience to sit and look at each one, you will be able to find even the rarest of mutations, provided that your butt can hold out. This is reminiscent of Rutherford’s atomic “bounce back” experiment, where the reco
il of alpha particles off of gold foil was so rare that it took many hours of watchful waiting, looking through a microscope in a dark room, to be able to measure them. Nonetheless, mutations, like alpha particle recoil, were considered so important to understanding the science that it was worth the wait.13
But Muller was frustrated with the very low frequencies of mutations in the flies.14 The rarity of mutations was slowing the pace of genetics research to a crawl. And it was sheer drudgery inspecting flies, one by one, looking for … what? Mutants could take any form, and screeners never knew exactly what body alterations to look for. If progress were to be made, the way they screened for mutants needed to change.
Then Muller had a brilliant idea. He knew (as did all other geneticists) that most mutations of genes are typically masked because chromosomes come in pairs (one coming from the father and one from the mother). Unless a gene undergoes a mutation on both chromosomes, the presence of a normal gene usually masks the mutant trait, because most mutations are recessive; hence, the mutation cannot be detected in the offspring. The exception to this is the X chromosome, the female sex chromosome. In females, the X chromosome is present as a pair, XX, the same as for the nonsex chromosomes. In males, however, it is accompanied by a male Y chromosome, forming a dissimilar XY pair. The Y is much smaller than the X chromosome, and only carries genes related to maleness, so there are no copies of X chromosome genes on the Y chromosome to mask X chromosome mutations.15 This makes the X chromosome an ideal target to screen for mutations. Even the recessive mutations on X should be revealed in male offspring because males only have one X chromosome, not two.
FIGURE 10.2. MALE FRUIT FLIES HAVE A SINGLE X CHROMOSOME. Similar to many other animal species, female fruit flies have two X chromosomes while males have only one. The key to Hermann Muller’s success in measuring radiation-induced mutations in fruit flies was his insight that this dissimilarity in the number of X chromosomes between genders meant that females had two copies of those genes located on the X chromosome. Muller was able to experimentally exploit this genetic gender difference by specifically studying X-linked lethal mutations of male flies. The increased sensitivity of his male fruit fly mutation assay allowed him to measure radiation mutagenesis down to very low doses, which had tremendous implications in terms of protection against radiation-induced cancer in humans.
The second part of Muller’s insight had to do with the type of mutant most likely to be produced. He reasoned that, since the X chromosome is relatively large, it likely carries many essential genes. Mutating an essential gene on an X chromosome should usually result in death of the offspring (i.e., an X-linked lethal mutation), but the female offspring would not die because they have a second X with a normal gene to mask the mutation. Males, in contrast, would get no such reprieve. For males, a mutation in an essential gene on their lone X chromosome would spell certain demise.
Putting this all together, it followed that in the broods of mated flies, where the mother passed down an X-linked lethal mutation, there should be missing males. That is, the ratio of males to females would not be the typical 1:1 sex ratio, since the males with the mutated X would not be viable. In those cases, the brood hatchlings would be predominantly females. With this realization, Muller had a screening test for X-linked lethal mutations that didn’t involve inspecting every fly with a jeweler’s magnifying glass, one at a time. In this new screening test, he could detect mutants simply by counting the females and males in a brood hatch and calculating the sex ratio. Broods with lower than 1:1 ratios (males to females) indicated an X-linked lethal mutation. Ironically, these postulated lethal mutations were not revealed by the presence of any mutants in the culture, but rather by their absence. The progeny of X-linked lethal mutations were male ghosts.
Muller’s sex-ratio assay amounted to a revolution in the way mutation screening was performed and quickened the pace of the work by orders of magnitude. By unmasking the X-linked lethal mutations, there were now mutation frequencies high enough to allow different chemical and physical agents to be tested to see how they modified the background mutation rates. This fast-paced approach had the potential to provide insight into the underlying mechanisms involved, which was always the fundamental goal of the research. Unfortunately, it would be six more years before Muller decided to test whether radiation could modify mutation frequencies. This is because one of the first sex-ratio screening experiments he performed suggested that radiation couldn’t be involved.
The first agent that Muller tested was temperature. He held flies at different temperatures and showed that the mutation rate modestly increased with temperature. The shape of the dose response curve revealed that the effect of temperature on mutation rate was not a straight line (i.e., linear), but rather was bent (i.e., curvilinear) in a way that suggested the relationship between temperature and mutation rates was logarithmic.16 Since it was well established that the rates of chemical reactions varied logarithmically with temperature, Muller reasoned that the underlying mechanism of mutation production in cells must, therefore, be chemically based, not physically based. So he proceeded to test various chemicals for their effects on mutation rates. He ignored radiation simply because it was a physical agent. What he did not realize at the time was that radiation interacts with the biochemistry of the cell.
Nonetheless, Muller eventually decided that it was a mistake to dismiss radiation without testing it. This was largely because it had previously been reported that radiation can damage chromosomes.17 Could something that damages chromosomes not have some effect on mutation rates? It didn’t seem likely, so Muller got a friend to set up an x-ray machine in his lab, and he used his sex-ratio screening test to measure mutation frequencies. What he found was astounding.
He started out with relatively high radiation doses but found that the radiation was causing sterility, so he wasn’t getting any offspring at all. Since there can’t be mutants if there are no offspring, he lowered the dose to just below the threshold for sterility in fruit flies.18 What he found was that this radiation dose increased the mutation rate to about 150 times the normal mutation rate!
Then he went further. He halved the dose and found half the number of mutants. When he halved the dose again, he again found half the mutants. In short, because Muller had so many flies, there was no radiation dose so low that he couldn’t detect some mutants, even when the frequency of mutations was extremely small. The sheer magnitude of the numbers of flies that he could irradiate and inspect allowed him to find mutations, regardless of how low the frequency. All he had to do was screen more flies.
The ability to detect mutations no matter how infrequently they occurred permitted him to test low doses of radiation that only rarely produced mutations. In the end, Muller concluded that there was some probability of producing inheritable mutations in fruit flies at all radiation doses, no matter how low, and that the frequency of mutations was directly proportional to dose (i.e., there was a linear dose response).19 Accordingly, there was no tolerance dose below which no mutations occurred! The armor of the concept of a tolerance dose for all health effects, which the radiation protection professionals relied on to provide absolute protection, had gotten its first chink. There would be more chinks to follow, but inheritable mutation production in fruit flies was the first. A paradigm shift had occurred in radiation biology, and everyone felt it.
DEAR COMRADE STALIN
Soon after Muller completed his radiation experiments, the Great Depression hit, and affected him particularly hard. He had struggled financially all of his life, and he had become greatly disillusioned with capitalism … and the United States. By this time, Muller was on the faculty of the University of Texas. Some of Muller’s students at Texas had introduced him to communism, and he helped them publish an underground student newspaper, The Spark, that promoted the virtues of communism to the wider university community. Always the enthusiast for social experimentation, Muller saw the burgeoning Soviet Union, and its great experiment wi
th communism, as the best hope for the world.
In 1933, Muller was invited to live and work in the Soviet Union by his Russian geneticist friend, Nikolai Ivanovich Vavilov (1887–1943). This was quite an honor, because Vavilov was among the most famous and respected scientists in the Soviet Union. So Muller enthusiastically immigrated to Moscow, where he thought he would find utopia. He took a position as senior geneticist at the Institute of Genetics of the USSR Academy of Sciences. By 1936, he felt confident enough in his position to write a letter directly to Soviet leader Joseph Stalin (1878–1953), to unfold his ideas for a biological revolution to augment the social revolution of communism. This biological revolution he termed “positive eugenics.” The plan called for the dedicated communist proletariat to forgo their own reproduction in favor of artificial insemination with sperm stock that was provided by the state and certified to have superior “genetic equipment.”
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