Daily Archives: January 16, 2017

“Mysterious” Non-protein-coding RNAs Play Important Roles In Gene Expression

In cells, DNA is transcribed into RNAs that provide the molecular recipe for cells to make proteins. Most of the genome is transcribed into RNA, but only a small proportion of RNAs are actually from the protein-coding regions of the genome.

“Why are the non-coding regions transcribed at all? Their function has been mysterious,” said Shelley Berger, PhD, a professor of Cell and Developmental Biology and director of the Penn Epigenetics Institute in the Perelman School of Medicine at the University of Pennsylvania.

Enhancer RNAs boost rate of gene expression from protein-coding gene.

Berger and Daniel Bose, PhD, a postdoctoral fellow in her lab, study the regulation of gene expression from enhancers, non–coding regions of the genome more distant from protein-coding regions. Enhancers boost the rate of gene expression from nearby protein-coding genes so a cell can pump out more of a needed protein molecule.  A mysterious subset of non-coding RNAs called enhancer RNAs (eRNAs) are transcribed from enhancer sequences. While these are important for boosting gene expression, how they achieve this has been completely unknown.

Shedding new light on these elusive eRNAs, they showed that CBP, an enzyme that activates transcription from enhancers, binds directly to eRNAs. This simple act controls patterns of gene expression in organisms by regulating acetylation, a chemical mark that directs DNA tightly packed in the nucleus of cells to loosen to promote transcription. Their findings are published this week in Cell.

“The cells in our bodies share the same genes and DNA sequences, and differ only in how these genes are expressed,” Bose said. “Enhancers and eRNAs are critical for this process. Our work shows an exciting new way that eRNAs produce these different patterns of gene expression. We asked if eRNAs work directly with CBP, and found that they do.”

Source: University of Pennsylvania

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How soil organisms can help plant species coexist

A new study of shrublands in Southwest Australia has identified how plant species can successfully coexist while competing for space and limited resources.

A team of international researchers, including Dr François Teste, Dr Michael Renton and Dr Etienne Laliberté from The University of Western Australia, found that part of the answer for high plant diversity is due to the many organisms that live in soils.

The study is the first of its kind to show that soil organisms interact with the way plants absorb nutrients which plays an important role in maintaining plant diversity in rich ecosystems.

Credit: The University of Western Australia

While some organisms are harmful to roots, causing diseases or even plant death, others are beneficial and enhance nutrient uptake and protect roots against harmful microorganisms.

Infertile shrublands in semi-arid regions support 20 per cent of the world’s plant species on just five per cent of the land surface, and those of Southwest Australia are some of the most species-rich of all.

By understanding the reason behind this high plant diversity, researchers can explore solutions for the conservation of species-rich ecosystems.

The study, published in the journal Science, looked at the interaction between a range of shrubland plant species and the organisms around their roots to understand how they influenced plants’ long-term coexistence.

Results reveal that soil organisms helped counteract growth differences between plant species. The researchers then used simulation modelling which showed this helped long-term coexistence of diverse plant species in ecosystems such as the Southwest Australian shrublands.

The researchers selected a large number of plant species and exposed them to soil organisms collected either from the plants own species or from other species.

While some plants grew better in their ‘own’ soils, many others actually performed best in the soils of other species.

Lead author Dr François Teste, said the results highlight the ‘unseen’ interactions that occur in soil.

“These interactions can enhance our understanding of the mechanisms that maintain local plant diversity, thus better informing ecosystem conservation efforts,” Dr Teste said.

“Ultimately, our study should enhance our ability to predict the way plants and ecosystems respond to global environmental change.”

Source: The University of Western Australia

Metabolic proteins relocate to jump-start an embryo’s genome

To turn on its genome — the full set of genes inherited from each parent — a mammalian embryo needs to relocate a group of proteins, researchers at the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA have discovered. The metabolic proteins, normally found in the energy-generating mitochondria of cells, move to the DNA-containing nuclei about two days after a mouse embryo is fertilized, according to the new study, led by senior author Utpal Banerjee.

Early in development, a mammalian embryo — or zygote — has all the materials it needs to grow and divide from genes and proteins that were contained in the egg cell. But after a few cell divisions, the zygote needs to activate its own genome. Researchers have never fully understood how this shift is made. They knew that certain metabolic compounds, such a pyruvate, were required, but had also observed that the mitochondria — which normally process pyruvate into energy — were small and inactive during this stage of development.

Protein coding genes are transcribed to an mRNA intermediate, then translated to a functional protein. RNA-coding genes are transcribed to a functional non-coding RNA. Credit: Thomas Shafee, Wikimedia Commons

Banerjee, a professor of molecular, cell, and developmental biology and co-director of the UCLA Broad Stem Cell Research Center, and colleagues confirmed that pyruvate was required for zygotes to activate their genomes by growing mouse zygotes in a culture dish lacking pyruvate. Then, in both mouse and human embryos, researchers used a number of methods to determine the location of proteins that process pyruvate through a metabolic program called the TCA cycle. Just before the embryos activated their genomes, the two-cell stage in mice, the TCA cycle proteins moved from the mitochondria to the nuclei of cells, the researchers discovered. While mouse cells grown in dishes lacking pyruvate normally stopped growing at the two-cell stage, the researchers could rescue these cells by adding a metabolic compound that’s produced by the TCA cycle. Repeating some of the experiments in human embryos, they confirmed that the metabolic proteins move from the mitochondria to the nucleus just as the genome is activated — at the six- to eight-cell stage for humans.

The importance of metabolic proteins to early embryonic development could affect future treatments for some types of infertility. In addition, the researchers hypothesize that some stem cells that have similar metabolic properties to early zygotes — including cancer stem cells — may relocate the TCA cycle proteins. Better understanding of the relocation could shed light on stem cell biology and alter cancer treatments.

Source: UCLA

Biofuel matchmaker: Finding the perfect algae for renewable energy

Streamlined process speeds up algae strain selection, could lower biofuel production costs

A dozen glass cylinders containing a potential payload of bright green algae are exposed to hundreds of multi-colored lights, which provide all of sunlight’s natural hues. The tiny LEDs brighten and dim to mimic the outdoors’ constantly changing conditions. To further simulate a virtual cloud passing overhead, chillers kick in and nudge the algae a little cooler.

A new, approximately $6-million collaborative project is using this unique climate-simulating laboratory system as part of a new streamlined process to quickly pare down heaps of algae species into just a few that hold the most promise for renewable fuels.

Discovering which algae species is best suited to make biofuel is no small task. Researchers have tried to evaluate algae in test tubes, but often find lab results don’t always mirror what happens when green goo is grown in outdoor ponds.

PNNL’s Laboratory Environmental Algae Pond Simulator system, also known as LEAPS, mimics the frequently shifting water temperatures and lighting conditions that occur in outdoor ponds at any given place on earth. This allows researchers to test multiple algae strains with the conditions at different places, but without the cost and time needed to actually grow them at those locations.

The Algae DISCOVR Project — short for Development of Integrated Screening, Cultivar Optimization and Validation Research — is trying out a new approach that could reduce the cost and the time needed to move promising algal strains from the laboratory and into production. At the end of the three-year pilot project, scientists hope to identify four promising strains from at least 30 initial candidates.

“Algae biofuel is a promising clean energy technology, but the current production methods are costly and limit its use,” said the project’s lead researcher, Michael Huesemann of the Department of Energy’s Pacific Northwest National Laboratory. “The price of biofuel is largely tied to growth rates. Our method could help developers find the most productive algae strains more quickly and efficiently.”

The project started this fall and is led by PNNL, out of its Marine Sciences Laboratory in Sequim, Washington. The project team includes three other DOE labs — Los Alamos National Laboratory, National Renewable Energy Laboratory and Sandia National Laboratories — as well as Arizona State University’s Arizona Center for Algae Technology and Innovation.

Algae can be turned into renewable biofuel, which is why scientists want to discover an inexpensive, fast-growing strain of algae. Scientists at Pacific Northwest National Laboratory have developed a system to speed up this search. The unique climate-simulating system uses temperature controls and multi-colored LED lights to mimic the constantly changing conditions of an outdoor algae pond. By simulating outdoor climates inside the lab, the system saves researchers time and expense.

Step by step

The project’s early work relies on PNNL’s Laboratory Environmental Algae Pond Simulator mini-photobioreactors, also known as LEAPS. The system mimics the frequently shifting water temperatures and lighting conditions that occur in outdoor ponds at any given place on earth. The system consists of glass column photobioreactors that act like small ponds and are placed in rows to allow scientists to simultaneously grow multiple different types of algae strains. Each row of LEAPS mini-photobioreactors is exposed to unique temperature and lighting regimens thanks to heaters, chillers and heat exchangers, as well as colored lights simulating the sunlight spectrum — all of which can be changed every second.

The first phase of the team’s multi-step screening process uses PNNL’s photobioreactors to cultivate all 30 strains under consideration and evaluate their growth rates. Algae strains with suitable growth will be studied further to measure their oil, protein and carbohydrate content, all of which could be used to make biofuels. The algae will also be tested for valuable co-products such as the food dye phycocyanin, which could make algae biofuel production more cost-effective. The first phase will also involve evaluating how resistant strains are to harmful bacteria and predators that can kill algae.

Next, the team will look for strains that produce 20 percent more biomass, or organic matter used to make biofuel, than two well-studied algae strains. The top-performing strains will then be sorted to find individual cells best suited for biofuel production, such as those that contain more oil. Those strains will also be exposed to various stresses to encourage rapid evolution so they can, for example, survive in the higher temperatures outdoor ponds experience in the summer.

Outside the box

After passing those tests, the remaining strains will be grown in large outdoor ponds in Arizona. Researchers will examine how algae growth in the outdoor ponds compares with the algal biomass output predicted in earlier steps. Biomass will also be harvested from outdoor-grown algae for future studies.

Finally, the team will further study the final algae strains that fare best outdoors to understand how fast they grow in different lighting and temperature conditions. That data will then be entered into PNNL’s Biomass Assessment Tool, which uses detailed data from weather stations and other sources to identify the best possible locations to grow algae. The tool will crunch numbers to help the team generate maps that illustrate the expected biomass productivity of each algae species grown in outdoor ponds at any location in the U.S.

Data and strains will be made public in the hopes that algae companies and other researchers will consider growing the most productive strains identified by the project.

This project is supported by DOE’s Office of Energy Efficiency and Renewable Energy.

Potential future work not included in the current project could include converting harvested algae into biofuels, examining operational changes such as crop rotation to further increase biomass growth, and assessing the technical feasibility and economic costs of making biofuel from algae selected through this process.

Source: PNNL

Study refutes how fruit flies developed alcohol tolerance

The common fruit fly, the tiny insect drawn to your beer or wine, has evolved to have an impressive tolerance for alcohol.

More than two decades ago, in one of the first papers using gene sequences to find signatures of natural selection, scientists hypothesized that a molecular change in an enzyme gave the Drosophila melanogaster fruit fly species its superior ability to metabolize alcohol. Scientists concluded that the change they found in the Alcohol dehydrogenase (ADH) protein could be the adaptation that allowed D. melanogaster to colonize ethanol-rich habitats in rotting fruit better than its nearly identical relative, Drosophila simulans.

Kristi Montooth, an associate professor in the School of Biological Sciences, displays a bottle containing fruit flies that are the subject of her research. She is a co-author of a new scientific paper investigating the evolutionary causes of fruit flies’ tolerance for alcohol. Image credit: Craig Chandler

It seemed a logical conclusion that the gene sequence changes that altered amino acids in an enzyme that breaks down alcohol would be the mechanism of natural selection.

However, the authors of a new paper published online Jan. 13 by Nature Ecology Evolution say they have now refuted that hypothesis.

Their findings indicate that intuition and signatures of selection in gene sequence may not be enough for scientists to conclusively solve the puzzles of molecular evolution. Tests also are needed to check how the changes function in organisms.

Using genetic engineering, scientists resurrected the fruit flies’ ADH protein from ancestral species to compare whether the amino acid changes that have occurred in D. melanogaster’s ADH enzyme actually improved the fruit flies’ ability to tolerate alcohol. The answer, conclusively, was “no.”

“This paper takes advantage of modern molecular biology and genetic approaches to test some of those hypotheses,” said University of Nebraska-Lincoln biologist Kristi L. Montooth, a fruit fly expert who co-authored the new study. “It doesn’t dispute that this species has a high ethanol tolerance. But it suggests that the molecular changes that have led to that high tolerance are not in this protein. They must be in other genes in the genome.”

The search for new pathways in fruit flies could offer new clues about alcohol tolerance in humans, Montooth added.

Montooth and University of Chicago researcher Mohammad A. Siddiq conducted experiments with living transgenetic flies in her Lincoln laboratory during October 2015. They found the ADH amino acid changes made no discernible difference in the species’ ability to survive while being fed increasingly heavy doses of alcohol. The flies had been genetically engineered by University of Wisconsin researcher David Loehlin so that their only genetic difference was in the ADH protein sequence.

The experiments with living fruit flies supported the findings of other experiments investigating the enzyme’s biochemistry.

With molecular evolution, scientists seek to identify the specific molecular changes underlying the trait changes that shape the evolutionary family tree.

“We’re generating so much sequence data right now, from so many species, that it’s relatively straightforward to look for signatures of selection in genes and to find good candidates for adaptations,” Montooth said. “But those are just candidates. You have to functionally test them if you want to say those are the variants favored by natural selection.”

Siddiq and Montooth began working together in 2008 after Siddiq took the freshman biology course Montooth taught while on faculty at Indiana University. He began working in her laboratory and co-authored research with her while he was an undergraduate student.

“Despite now being in my fifth year of graduate school, I’m still publishing papers with the professor that I met on my first day of college,” Siddiq said.

Siddiq, who is the study’s first author, is now pursuing a doctoral degree at the University of Chicago, where he is studying with co-author Joseph W. Thornton, a professor who studies the molecular mechanisms of evolution. David W. Loehlin, a post-doctoral researcher at the University of Wisconsin-Madison, also participated in the study, creating the transgenic flies used in the experiments.

Siddiq came to Lincoln to work with Montooth on the fruit fly study, he said, because of her experience and expertise in evolutionary physiology and because of her influence on his career.

“I took her introductory biology class (as a college freshman) and loved it because her passion for evolution and its role as a unifying principle in biology really came through,” Siddiq said. “My appreciation for studying traits not only in the test tube, but in the level of organisms, comes from the experiments I did during my undergraduate years. “

The new study is the first demonstration of an approach combining ancestral sequence reconstruction with the making of transgenic organisms to directly test hypotheses at multiple biological levels, Siddiq said. It’s a broadly applicable approach that in theory could be used with other organisms that lend themselves to transgenic engineering.

“One of the neat things about this paper is that it looks at the effects of this gene’s mutations at the level of how the enzyme functions in the test tube, how it functions in the fly and then the consequences for how the fly deals with ethanol,” Montooth said. “It’s looking at effects of mutations across different levels of biological organization. Each of the co-authors played a role by investigating a different level of organization.”

The study received funding through the National Science Foundation and the National Institutes of Health, including an NIH training grant and NSF fellowship for Siddiq and an NSF CAREER award for Montooth. Loehlin received support from a Howard Hughes Medical Institute postdoctoral fellowship.

Source: University of Nebraska-Lincoln