From 1997 to 2014, the U.S. Fish and Wildlife Service moved more than 9,100 Mojave desert tortoises to the 100-square-kilometer (about 39 miles square) Large Scale Translocation Site. The newcomers, many of which were abandoned pets or had been displaced by development, joined nearly 1,500 desert tortoises already living there.

Conventional wisdom would suggest that tortoises from areas closest to the translocation site would fare best. But a new UCLA study, published today in Science, found no connection between the tortoises’ place of origin and their chances of survival. It did, however, uncover a far better predictor.

Tortoises with lots of genetic variation were much more likely to survive after their relocation, said UCLA conservation ecologist Brad Shaffer, the study’s senior author. Like most organisms, tortoises have two copies of their entire genome, with one copy from each parent. The more those copies differ from each other, the higher the organism’s heterozygosity.

The researchers compared translocated tortoises that lived or died over the same time period after being relocated to the site. They found that survivors averaged 23% greater heterozygosity than those that perished. Simply put, tortoises with more genetic variation had higher survival rates.

“It flies in the face of what we know from other translocation studies, but lots of genetic variation was hands-down the best predictor of whether a tortoise lived or died,” said Shaffer, a professor of ecology and evolutionary biology and director of the UCLA La Kretz Center for California Conservation Science. “Relocating endangered plants and animals is increasingly necessary to counteract the effects of climate change, and this gives us a new tool to increase survival rates.”

Although the relationship between heterozygosity and survival was well supported by the study, it’s unclear why greater genetic variation is linked to survival rates, said former UCLA postdoctoral scholar Peter Scott, the study’s lead author.

“Potentially, individuals with higher heterozygosity have more genomic flexibility,” said Scott, who is now an assistant professor at West Texas A&M University. “It’s likely that tortoises with more variation have a better chance of having one copy of a gene that works really well in stressful or new environments compared to those individuals with two identical copies that only work really well in their environment of origin.”

The researchers wanted to make tortoise conservation efforts more effective, and uncover trends that would help other species as well, Scott said.

“Oftentimes, the chances of success for relocating plants or animals is pretty dismal,” he said. “We wanted to understand why, and use that understanding to increase survival.”

Over the years, tortoises that were given up as pets, or removed from places like developments in suburban Las Vegas and solar farms in the desert, were surrendered to the U.S. Fish and Wildlife Service.

The agency took blood samples to screen for diseases and marked each animal before releasing them into the Ivanpah Valley site, which enabled the animals to be tracked in later surveys. The UCLA researchers sequenced blood samples drawn from 79 tortoises that were released to the site and were known to be alive in 2015, and from another 87 known to have died after they were released at the site.

Although the Large Scale Translocation Site provided an intriguing dataset, it’s not the same as a controlled experiment. Additional studies would be needed to understand why more heterozygous tortoises had a higher survival probability and precisely how much of an increase in genetic variation improves a tortoise’s odds of surviving.

“The only reason we could do this study was because the U.S. Fish and Wildlife Service was incredibly forward-thinking when they set up the translocation site and tracked who lived and died,” Shaffer said. “Many died, and no one was happy about that. But we can learn a lot from that unfortunate result to help conservation management improve.

“When thinking about moving animals or plants out of danger, or repopulating an area emptied by wildfire, now we can easily and economically measure genetic variability to better gauge the survival probability of those translocated individuals. It’s not the only criteria, but it’s an important piece of the puzzle.”

The finding concerns a key process essential to life: the transcription phase of gene expression, which enables cells to live and do their jobs.

During transcription, an enzyme called RNA polymerase wraps itself around the double helix of DNA, using one strand to match nucleotides to make a copy of genetic material — resulting in a newly synthesized strand of RNA that breaks off when transcription is complete. That RNA enables production of proteins, which are essential to all life and perform most of the work inside cells.

Just as with any coherent message, RNA needs to start and stop in the right place to make sense. A bacterial protein called Rho was discovered more than 50 years ago because of its ability to stop, or terminate, transcription. In every textbook, Rho is used as a model terminator that, using its very strong motor force, binds to the RNA and pulls it out of RNA polymerase. But a closer look by these scientists showed that Rho wouldn’t be able to find the RNAs it needs to release using the textbook mechanism.

“We started studying Rho, and realized it cannot possibly work in ways people tell us it works,” said Irina Artsimovitch, co-lead author of the study and professor of microbiology at The Ohio State University.

The research, published online by the journal Science today, Nov. 26, 2020, determined that instead of attaching to a specific piece of RNA near the end of transcription and helping it unwind from DNA, Rho actually “hitchhikes” on RNA polymerase for the duration of transcription. Rho cooperates with other proteins to eventually coax the enzyme through a series of structural changes that end with an inactive state enabling release of the RNA.

The team used sophisticated microscopes to reveal how Rho acts on a complete transcription complex composed of RNA polymerase and two accessory proteins that travel with it throughout transcription.

“This is the first structure of a termination complex in any system, and was supposed to be impossible to obtain because it falls apart too quickly,” Artsimovitch said.

“It answers a fundamental question — transcription is fundamental to life, but if it were not controlled, nothing would work. RNA polymerase by itself has to be completely neutral. It has to be able to make any RNA, including those that are damaged or could harm the cell. While traveling with RNA polymerase, Rho can tell if the synthesized RNA is worth making — and if not, Rho releases it.”

Artsimovitch has made many important discoveries about how RNA polymerase so successfully completes transcription. She didn’t set out to counter years of understanding about Rho’s role in termination until an undergraduate student in her lab identified surprising mutations in Rho while working on a genetics project.

Rho is known to silence the expression of virulence genes in bacteria, essentially keeping them dormant until they’re needed to cause infection. But these genes do not have any RNA sequences that Rho is known to preferentially bind. Because of that, Artsimovitch said, it has never made sense that Rho looks only for specific RNA sequences, without even knowing if they are still attached to RNA polymerase.

In fact, the scientific understanding of the Rho mechanism was established using simplified biochemical experiments that frequently left out RNA polymerase — in essence, defining how a process ends without factoring in the process itself.

In this work, the researchers used cryo-electron microscopy to capture images of RNA polymerase operating on a DNA template in Escherichia coli, their model system. This high-resolution visualization, combined with high-end computation, made accurate modeling of transcription termination possible.

“RNA polymerase moves along, matching hundreds of thousands of nucleotides in bacteria. The complex is extremely stable because it has to be — if the RNA is released, it is lost,” Artsimovitch said. “Yet Rho is able to make the complex fall apart in a matter of minutes, if not seconds. You can look at it, but you can’t get a stable complex to analyze.”

Using a clever method to trap complexes just before they fall apart enabled the scientists to visualize seven complexes that represent sequential steps in the termination pathway, starting from Rho’s engagement with RNA polymerase and ending with a completely inactive RNA polymerase. The team created models based on what they saw, and then made sure that these models were correct using genetic and biochemical methods.

Though the study was conducted in bacteria, Artsimovitch said this termination process is likely to occur in other forms of life.

“It appears to be common,” she said. “In general, cells use similar working mechanisms from a common ancestor. They all learned the same tricks as long as these tricks were useful.”

Artsimovitch, working with an international research team of collaborators, co-led the study with Markus Wahl, a former Ohio State graduate student now at Freie Universität Berlin.

This work was supported by grants from the German Research Foundation; the German Federal Ministry of Education and Research; the Indian Council of Medical Research; the Department of Biotechnology, Government of India; the National Institutes of Health; and the Sigrid Jusélius Foundation.

When a slow-speed, high-power laser is melting metal powder during the 3D printing of a part, a keyhole-shaped cavity in the melt pool can result. Pores, i.e. defects, form at the bottom of the keyhole. New research published in Science reveals how the pores are generated and become defects trapped in solidifying metal.

“The real practical value of this research is that we can be precise about controlling the machines to avoid this problem,” says Anthony D. Rollett, a professor of materials science and engineering in Carnegie Mellon College of Engineering and a lead co-author of the paper, “Critical instability at moving keyhole tip generates porosity in laser melting.”

Building on previous research that quantified the keyhole phenomenon, the research team used extremely bright high-energy x-ray imagining to watch instabilities of the keyhole. Pores form during fluctuations of the keyhole, and it changes its shape: the keyhole tip morphs into a “J” shape and pinches off. This unstable behavior generates acoustic waves in the liquid metal that force the pores away from the keyhole so that they survive long enough to get trapped in the resolidifying metal. The team is the first to focus on this behavior and identify what is happening.

“When you have a deep keyhole, the walls oscillate strongly. Occasionally, the oscillations are strong enough at the bottom of the keyhole that they pinch off, leaving a large bubble behind. Sometimes this bubble never reconnects to the main keyhole. It collapses and generates an acoustic shock wave. This pushes the remaining pores away from the keyhole,” explains Rollett.

It’s important to note that keyholes themselves are not flaws and, e.g., they increase the efficiency of the laser. Using synchrotron x-ray equipment at Argonne National Laboratories, the only facility in the United States where the researchers could run these experiments, they noted that there is a well-defined boundary between stable versus unstable keyholes.

“As long as you stay out of the danger zone [i.e., too hot, too slow], the risk of leaving defects behind is quite small,” says Rollett.

Fluctuations in the keyhole’s depth increase strongly with decreasing scan speed and laser power on the unstable side of the boundary.

“You can think of the boundary as a speed limit, except it is the opposite of driving a car. In this case, it gets more dangerous as you go slower. If you’re below the speed limit, then you are almost certainly generating a defect,” adds Rollett.

At a broader scale, by proving the existence of well-defined keyhole porosity boundaries and demonstrating the ability to reproduce them, science can offer a more secure basis for predicting and improving printing processes. Rollett, who is the faculty co-director of Carnegie Mellon’s Next Manufacturing Center, thinks that the findings from this research will quickly find their way into how companies operate their 3D printers.

Watch Prof. Rollett discuss the process of 3D printing with metals as well as using advanced characterization techniques to solve industry challenges in the field.

The research team includes co-lead author Tao Sun, University of Virginia; Cang Zhao, Tsinghua University, China; Niranjan D. Parab and Kamel Fezzaa, Argonne National Laboratory; Xuxiao Li and Wenda Tan, University of Utah.

Funding sources include the Department of Energy, the Department of Defense, the National Aeronautics and Space Administration (NASA) University Leadership Initiative program, the National Science Foundation, and startup funds from the Department of Mechanical Engineering at Tsinghua University and the University of Virginia.