Scientists engineer animals with ancient genes to test causes of evolution

A transgenic fruit fly engineered to carry the alcohol dehydrogenase gene as it existed about 4 million years ago. Thousands of these 'ancestralized' flies were bred and studied for their ability to metabolize alcohol and to survive on an.

Scientists at the University of Chicago have created the first genetically modified animals containing reconstructed ancient genes, which they used to test the evolutionary effects of genetic changes that happened in the deep past on the animals' biology and fitness.

The research, published early online in Nature Ecology & Evolution on Jan. 13, is a major step forward for efforts to study the genetic basis of adaptation and evolution. The specific findings, involving the fruit fly's ability to break down alcohol in rotting fruit, overturn a widely-held hypothesis about the molecular causes of one of evolutionary biology's classic cases of adaptation.



"One of the major goals of modern evolutionary biology is to identify the genes that caused species to adapt to new environments, but it's been hard to do that directly, because we've had no way to test the effects of ancient genes on animal biology," said Mo Siddiq, a graduate student in the Department of Ecology and Evolution at the University of Chicago, one of the study's lead scientists.

"We realized we could overcome this problem by combining two recently developed methods—statistical reconstruction of ancient gene sequences and engineering of transgenic animals," he said.

Until recently, most studies of molecular adaptation have analyzed gene sequences to identify "signatures of selection"—patterns suggesting that a gene changed so quickly during its evolution that selection is likely to have been the cause. The evidence from this approach is only circumstantial, however, because genes can evolve quickly for many reasons, such as chance, fluctuations in population size, or selection for functions unrelated to the environmental conditions to which the organism is thought to have adapted.

Siddiq and his advisor, Joe Thornton, PhD, professor of ecology and evolution and human genetics at the University of Chicago, wanted to directly test the effects of a gene's evolution on adaptation. Thornton has pioneered methods for reconstructing ancestral genes—statistically determining their sequences from large databases of present-day sequences, then synthesizing them and experimentally studying their molecular properties in the laboratory. This strategy has yielded major insights into the mechanisms by which biochemical functions evolve.

Thornton and Siddiq reasoned that by combining ancestral gene reconstruction with techniques for engineering transgenic animals, they could study how genetic changes that occurred in the deep past affected whole organisms-their development, physiology, and even their fitness.



"This strategy of engineering 'ancestralized animals' could be applied to many evolutionary questions," Thornton said. "For the first test case, we chose a classic example of adaptation-how fruit flies evolved the ability to survive the high alcohol concentrations found in rotting fruit. We found that the accepted wisdom about the molecular causes of the flies' evolution is simply wrong."

The fruit fly Drosophila melanogaster is one of the most studied organisms in genetics and evolution. In the wild, D. melanogaster lives in alcohol-rich rotting fruit, tolerating far higher alcohol concentrations than its closest relatives, which live on other food sources. Twenty-five years ago at the University of Chicago, biologists Martin Kreitman and John McDonald invented a new statistical method for finding signatures of selection, which remains to this day one of the most widely used methods in molecular evolution. They demonstrated it on the alcohol dehydrogenase (Adh) gene—the gene for the enzyme that breaks down alcohol inside cells—from this group of flies. Adh had a strong signature of selection, and it was already known that D. melanogaster flies break down alcohol faster than their relatives. So, the idea that the Adh enzyme was the cause of the fruit fly's adaptation to ethanol became the first accepted case of a specific gene that mediated adaptive evolution of a species.

Siddiq and Thornton realized that this hypothesis could be tested directly using the new technologies. Siddiq first inferred the sequences of ancient Adh genes from just before and just after D. melanogaster evolved its ethanol tolerance, some two to four million years ago. He synthesized these genes biochemically, expressed them, and used biochemical methods to measure their ability to break down alcohol in a test tube.



The results were surprising: the genetic changes that occurred during the evolution of D. melanogaster had no detectable effect on the protein's function.
Working with collaborators David Loehlin at the University of Wisconsin and Kristi

Montooth at the University of Nebraska, Siddiq then created and characterized transgenic flies containing the reconstructed ancestral forms of Adh. They bred thousands of these "ancestralized" flies, tested how quickly they could break down alcohol, and how well the larvae and adult flies survived when raised on food with high alcohol content. Surprisingly, the transgenic flies carrying the more recent Adh were no better at metabolizing alcohol than flies carrying the more ancient form of Adh. Even more strikingly, they were no better able to grow or survive on increasing alcohol concentrations. Thus, none of the predictions of the classic version of the story were fulfilled. There is no doubt that D. melanogaster did adapt to high-alcohol food sources during its evolution, but not because of changes in the Adh enzyme.

"The Adh story was accepted because the ecology, physiology, and the statistical signature of selection all pointed in the same direction. But three lines of circumstantial evidence don't make an airtight case," Thornton said. "That's why we wanted to test the hypothesis directly, now that we finally have the means to do so."

Siddiq and Thornton hope that the strategy of making ancestralized transgenic will become the gold standard in the field to decisively determine the historical changes in genes to their changes on organisms' biology and fitness.

For his part, Kreitman, who is still a professor of ecology and evolution at UChicago, has been supportive of the new research, helping advise Siddiq on the project and sharing his vast knowledge about molecular evolution and Drosophila genetics.



"From the beginning, Marty was excited about our experiments, and he was just as supportive when our results overturned well-known conclusions based on his past work," Siddiq said. "I think that's extremely inspiring."

Source: University of Chicago, Medical Center

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How Life began? A New Discovery in Astrobiology

The Ribonucleic Acid, vital element for living, is product of evolution and a “Mother” molecule may help give birth to the RNA.

All life on Earth uses DNA to encode and store genes, and to pass them on from one generation to the next. RNA, a close molecular relative of DNA, is used within living cells to carry out a wide variety of important biological functions, including protein synthesis.

DNA and RNA are both extremely complex molecules, a fact that has fueled a long-standing debate about the origins of life. RNA looks potentially older and more versatile than DNA so many scientists believe that RNA came before DNA. But did life begin with RNA, or was there something even simpler that preceded it?

Scientists have found it difficult to deduce the chemical environment on early Earth when life began. Nevertheless, there have been a number of notable successes in this area of research, including the discovery of reactions that produce the building blocks of RNA. However, a reaction that produces actual polymers of RNA has not been found, causing many scientists to conclude that RNA is the product of evolution.

“The origin of RNA is something I’ve been working on for two decades,” said Nicholas Hud, head of the Georgia Institute of Technology’s Center for Chemical Evolution, where researchers are attempting to figure out how life began. “It is easy to speculate that some other molecule came before RNA, but determining the structure of molecules that might actually have come before RNA is a major challenge for chemists.”



A recent paper by Hud and his team found that pro-flavine, a small planar molecule that binds between adjacent base pairs of a DNA, can dramatically increase the stability of DNA or RNA in the lab when single strands of the complex molecules are synthetically bound to an unnatural nucleic acid. (“Unnatural” means that these are molecules similar to natural DNA or RNA, but with different backbone structures.)

“Proflavine can cause the formation of a duplex from two nucleic acids that otherwise do not form duplexes at all,” Hud said.
He added that their results are important for two reasons. The first is that duplexes formed by DNA and an unnatural nucleic acid could be used to create dynamic structures that assemble only when proflavine, or a similar molecule, is present. This property has potential applications in nanotechnology, or the technology of very small things.

A second and more fundamental implication of this work has to do with our understanding of the “RNA World” hypothesis, the theory that RNA was the first molecule of life. After decades of unsuccessful attempts to create RNA in model prebiotic (non-biological) reactions, many chemists that study the origin of life believe that there must have been some other RNA-like polymer before RNA. Hud said the first of these polymers could be called a ‘proto-RNA‘, and each evolutionary step between proto-RNA and current RNA is a ‘pre-RNA.’

“There have been many proposals for possible pre-RNA structures,” he said. “While it may not be possible to determine the exact structure of any of the molecules that served as the ancestors of RNA, researchers generally agree that the ability to form a duplex with RNA is an important criterion that must be met for a molecule to be considered a possible ancestor of RNA.”



Back in 2000 Hud and his collaborator Frank Anet proposed that small molecules might have helped the synthesis of RNA, or the original ancestor of RNA, if RNA came later. They called these hypothetical small molecules ‘molecular midwives,’ to reflect that these molecules would have helped give birth to RNA. They proposed that molecular midwives would have been similar to molecules that are currently known to bind DNA and RNA, such as fluorescent dye molecule known as “intercalators” that are used to visualize DNA in the laboratory.

Most recently, Hud and his collaborators have discovered that proflavine, a particular intercalator, is able to cause the formation of a double helix, like that formed by two DNAmolecules, but between RNA and a non-natural RNA-like molecule that has a “backbone” structure distinct from RNA and DNA. In the absence of proflavine these molecules do not form double helixes.

This result has important implications regarding the origin of the first RNA-like molecules of life. In particular, the number of molecules that could have served as the first genetic molecules of life might have been much greater than previously expected if intercalator molecules we present on the early Earth. Hud notes that proflavine is not a potentially prebiotic molecule, but was used in their study as a model for the type of intercalator molecules that may have been available on early Earth.

Hud has spent years studying the potential role of intercalators in the origin of RNA. Very few studies have investigated the ability of intercalators to bind non-natural RNA-like molecules, and no previous studies had investigated the possibility that intercalators might allow RNA to pair with polymers that would otherwise be “incompatible” with RNA.

Connections made within the Center for Chemicals Evolution (or CCE) allowed these studies to move to a whole new level. Ram Krishnamurthy, a fellow member of the CCE who is at The Scripps Research Institute in La Jolla, Calif., was synthesizing with his co-workers a polymer called ISO-GNA that is in some ways simpler than RNA, and they thought this polymer might shed light on the structural requirements for the simplest informational system RNA.

Their observation that ISO-GNA has limited base-pairing with RNA made them question the currently accepted requirements for ancestors of RNA. Conversations within the CCE resulted in Hud’s group testing the ability of an intercalator to facilitate the pairing of Krishnamurthy’s molecule (ISO-GNA) with RNA.



“In our earlier work on iso-GNA revealed that this molecule did not form duplexes that are stable as those of DNA or RNA,” Krishnamurthy said. “We proposed that this limitation of ISO-GNA could be overcome by the use of intercalators since they are known to stabilize base-pairing within a duplex structure by enhancing base-stacking. What is fascinating in this study is that the increase we observed for the stability of iso-GNA duplexes in the presence of proflavine is much larger than the increased stability those observed when proflavine binds DNA or RNA, far exceeding our expectations. This study should allow for a greater flexibility when searching for possible prebiotic polymers that are able to interact with RNA (or DNA).”

These latest results have caused Hud and Krishnamurthy to broaden their view of which molecules might have come before RNA. These two researchers and their co-workers are currently trying to find a possible ancestor of RNA that is able to spontaneously form from molecules that were present on the early Earth. They remain more open than ever to the idea that some molecules not seen in life today may have been necessary to get life started, perhaps molecules that we could view as the “midwives” that helped give “birth” to RNA.



Funding for the research was provided by the National Science Foundation (which funds CCE more generally) and support from the NASA Astrobiology Program at NASA.

Source: Aaron Gronstal, Astrobio.net

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