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  • richardmitnick 12:13 pm on August 23, 2015 Permalink | Reply
    Tags: , Biology, ,   

    From Scripps: “Hands-On Research 101: Internships Introduce Undergrads to Biomedical Science in Action” 


    Scripps Research Institute

    August 24, 2015
    Madeline McCurry-Schmidt

    SURF Intern Joshua David says the internship at TSRI gave him new opportunities to learn about biomedicine. (Photo by Cindy Bruaer.)

    When Joshua David saw scientists from The Scripps Research Institute (TSRI) discussing Ebola virus research on the news last year, he wanted to help.

    “I discovered that Scripps is one of the top places looking at Ebola virus at the molecular level,” said David, an undergraduate chemistry major at Virginia Commonwealth University. “The scientists at Scripps are trying to help people who are suffering and dying right now.”

    David quickly got in touch with Ebola researchers at TSRI and learned about the institute’s Summer Undergraduate Research Fellows (SURF) Program, organized by the TSRI Office of Graduate Studies. The SURF Program is a 10-week internship program at TSRI that has brought 38 undergraduates to TSRI’s California and Florida campuses this year. It’s one of several outreach programs, including a summer high school internship program where another 30 students work side-by-side with researchers.

    As a SURF intern, David flew into San Diego in June and spent his summer in Associate Professor Andrew Ward’s lab.

    Learning New Techniques

    David said the internship gave him new opportunities to learn about biomedicine.

    “I’m very interested in structural biology and virology; however, these courses are not offered at my university,” David explained. “Coming here is a great opportunity because it allows me learn techniques used in these fields and gain general knowledge of each field in the process.”

    Under the guidance of C. Daniel Murin, a graduate student in the Ward lab, David learned how to build 3-D structures of proteins involved in Ebola virus attacks. The SURF program emphasizes hands-on research, so David learned to use a technique called electron microscopy (EM) to study exactly how Ebola virus interacts with antibodies.

    “I wanted to take him through that process, so he can go through it almost independently by the end of the summer,” said Murin.

    David worked with Murin on several projects, including studies involving the experimental Ebola virus treatment ZMapp, which has also been the topic of previous studies at TSRI.

    David said one challenge this summer was tackling how to use a molecular imaging program necessary for research with EM.

    “Then I just had to sit down and figure it out,” he said. “It took me about eight hours, but now I understand how to do it.”

    Helping Patients

    David hopes to bring together research and patient care in a future career as a physician-scientist. As a high school student, David interned in a hospital’s intensive care unit. He watched as patients succumbed to diseases like acute respiratory distress syndrome (ARDS)—where doctors have few treatments to offer.

    A technique like EM could give David and other scientists a better look at the proteins involved in disease—from Ebola to ARDS—and lead to new treatments.

    “You can understand how things work in cells at the atomic level, and that really interests me,” said David.

    Before David headed back to Virginia at the end of the summer, he presented a poster outlining his work to peers and supervisors at TSRI. It was chance to show what he’s learned—and why he wants to be part of the next generation of scientists.

    About the Summer Undergraduate Research Fellows (SURF) Program

    TSRI’s 10-week SURF program provides participants the opportunity to perform cutting-edge research in one of 250 laboratories side-by-side with TSRI’s world-renowned faculty. The goals of the program are to:

    Make program participants feel comfortable in a lab setting and increase their research skills
    Teach participants to think critically about the theory and application of biomedical research
    Increase the participants’ proficiency in communicating scientific concepts
    Increase the number of underrepresented and first-generation to college students who consider careers in biomedical research.

    Students can choose to apply to either the La Jolla campus in California or the Jupiter campus in Florida. Learn more at TSRI’s Education website.

    See the full article here.

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    The Scripps Research Institute (TSRI), one of the world’s largest, private, non-profit research organizations, stands at the forefront of basic biomedical science, a vital segment of medical research that seeks to comprehend the most fundamental processes of life. Over the last decades, the institute has established a lengthy track record of major contributions to the betterment of health and the human condition.

    The institute — which is located on campuses in La Jolla, California, and Jupiter, Florida — has become internationally recognized for its research into immunology, molecular and cellular biology, chemistry, neurosciences, autoimmune diseases, cardiovascular diseases, virology, and synthetic vaccine development. Particularly significant is the institute’s study of the basic structure and design of biological molecules; in this arena TSRI is among a handful of the world’s leading centers.

    The institute’s educational programs are also first rate. TSRI’s Graduate Program is consistently ranked among the best in the nation in its fields of biology and chemistry.

  • richardmitnick 10:49 pm on August 12, 2015 Permalink | Reply
    Tags: , Biology, , Flippases, Lipids   

    From ETH: “How lipids are flipped” 

    ETH Zurich bloc

    ETH Zurich

    Peter Rüegg

    Comparison of cavities (green) in inward-facing (left) and outward-occluded (right) states of PglK, with native LLO (middle) shown as space-filling model for size reference. (Illustration: from Perez et al, 2015)

    A team of researchers at ETH Zurich and the University of Bern has succeeded in determining the structure of a lipid flippase at high resolution, which has provided insight into how this membrane protein transports lipids by flipping.

    Biological membranes have a fundamental role in separating the interior of cells from the extracellular space and in helping determine cellular shape and size. They consist of a double layer (“bilayer”) of lipids that contain a hydrophilic head group and generally two long, hydrophobic tails. Whereas the head groups face outwards, the hydrophobic tail face each other. Numerous other components are embedded in membranes, including pore-forming proteins and transport proteins.

    Countless vital processes occur at membranes, including the transport of various substances. The transport of phospholipids and lipid-linked oligosaccharides (LLO) is particularly difficult to achieve due to the bipolar nature of the lipid bilayer – hydrophobic interior, hydrophilic surface. This is why flippases are required to transport lipids from one side of the membrane to the other, essentially flipping their orientation. Flippases have important roles in maintaining the asymmetry of cellular membranes, i.e. in the different lipid composition of the outer and inner sides. In mammals, this affects various processes such as blood coagulation, immune recognition and programmed cell death, or apoptosis. Flippases are also essential for transporting lipid-linked oligosaccharides (“LLOs”), which are transferred onto acceptor proteins during N-linked protein glycosylation.

    Flippase structure revealed for the first time

    Until now biologists knew neither the high-resolution structures of flippases nor the exact mechanism used to flip LLOs. Three research teams from ETH Zurich and the University of Bern, led by ETH professor Kaspar Locher, have now determined the structure of one such flippase, the PglK protein from the bacterium Campylobacter jejuni. The study has been published in the journal Nature.

    This required the researchers to purify the flippase from bacterial membranes and generate three-dimensional crystals, which were then analysed using X-ray crystallography to determine the positions of all atoms. The scientists determined three distinct structures that corresponded to different states of the flippase during the reaction. Their data allowed the researchers to deduce a molecular mechanism of how PglK flips LLOs.

    The researchers show that PglK consists of two identical subunits that move like a pair of scissors when energy (ATP) is consumed. Similar to a credit card reader, the oligosaccharide moiety of the lipid-linked oligosaccharide is then pulled into a hydrophilic channel within the flippase. The hydrophobic, lipidic tail of the LLO molecule remains exposed to the lipidic membrane, causing the LLO to change its orientation so that the sugar part ends up on the outside of the membrane. The flippase itself does not change its orientation during translocation reaction and therefore acts as a catalyst.

    Fundamentally different mechanism

    The newly-discovered mechanism fundamentally differs from previously known transport processes that catalyze import or export of soluble substrates. “The flipping of lipids in membranes has always fascinated biochemists and cell biologists; the biological solution to this problem thrills us!” says co-author Markus Aebi, Professor of Microbiology at ETH Zurich.

    The research groups from ETH Zurich and the University of Bern are the first to have solved the fundamental biological puzzle of how LLOs are flipped. They developed a novel method for studying the reaction in vitro. ETH Professor Aebi insists that only through the cooperation of structural biologists, chemists and microbiologists was it possible to decipher this basic mechanism. “Every group brought their own expertise from their respective fields. This was the only way we could succeed.”

    Therapeutic applications?

    Although the present work constitutes basic research, there are diseases associated with mutations in a human flippase, explains Aebi. These diseases are termed ‘congenital disorders of glycosylation’. More than 10,000 glycosylation sites in various proteins have been identified in humans, “which is why changes in glycosylation in which flippase plays a crucial role affect so many processes in the body,” says the ETH professor. Examples of this include the development and maturation of the central nervous system.

    Whether the newly acquired knowledge of the bacterial flippase PglK leads to novel therapeutic approaches is unclear at present. However, flippases already form an essential part of biotechnological systems that are used to generate glycoproteins desigend for various uses in diagnostics and potential therapeutic agents.

    LLO synthesis and N-glycosylation in Campylobacter jejuni at a glance: The LLO’s lipidic tail is shown in purple. A polypeptide chain is shown in blue. PglH (red) is the glycosyltransferace that couples the oligosaccharide to the polypeptide chain.


    Perez C, Gerber S, Boilevin J, Bucher M, Darbre T, Aebi M, Reymond J-L, Locher KP. Molecular view of lipid-linked oligosaccharide translocation across biological membranes. Nature, Advanced online publication, 12th August 2015. DOI: 10.1038/nature14953

    See the full article here.

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    ETH Zurich campus
    ETH Zurich is one of the leading international universities for technology and the natural sciences. It is well known for its excellent education, ground-breaking fundamental research and for implementing its results directly into practice.

    Founded in 1855, ETH Zurich today has more than 18,500 students from over 110 countries, including 4,000 doctoral students. To researchers, it offers an inspiring working environment, to students, a comprehensive education.

    Twenty-one Nobel Laureates have studied, taught or conducted research at ETH Zurich, underlining the excellent reputation of the university.

  • richardmitnick 11:49 am on August 4, 2015 Permalink | Reply
    Tags: , Biology,   

    From LBL: “Atomic View of Microtubules” 

    Berkeley Logo

    Berkeley Lab

    August 4, 2015
    Lynn Yarris (510) 486-5375

    Microtubules are hollow cylinders with walls made up of tubulin proteins – alpha (green) and beta (blue) – plus EB proteins (orange) that can either stabilize or destabilize the structure of the tubulin proteins.

    Microtubules, hollow fibers of tubulin protein only a few nanometers in diameter, form the cytoskeletons of living cells and play a crucial role in cell division (mitosis) through their ability to undergo rapid growth and shrinkage, a property called “dynamic instability.” Through a combination of high-resolution cryo-electron microscopy (cryo-EM) and a unique methodology for image analysis, a team of researchers with Berkeley Lab and the University of California (UC) Berkeley has produced an atomic view of microtubules that enabled them to identify the crucial role played by a family of end-binding (EB) proteins in regulating microtubule dynamic instability.

    During mitosis, microtubules disassemble and reform into spindles that are used by the dividing cell to move chromosomes. For chromosome migration to occur, the microtubules attached to them must disassemble, carrying the chromosomes in the process. The dynamic instability that makes it possible for microtubules to transition from a rigid polymerized or “assembled” nucleotide state to a flexible depolymerized or “disassembled” nucleotide state is driven by guanosine triphosphate (GTP) hydrolysis in the microtubule lattice.

    “Our study shows how EB proteins can either facilitate microtubule assembly by binding to sub-units of the microtubule, essentially holding them together, or else cause a microtubule to disassemble by promoting GTP hydrolysis that destabilizes the microtubule lattice,” says Eva Nogales, a biophysicist with Berkeley Lab’s Life Sciences Division who led this research.

    Gregory Alushin and Eva Nogales studying images of microtubule structures. (Photo by Roy Kaltschmidt)

    Nogales, who is also a professor of biophysics and structural biology at UC Berkeley and investigator with the Howard Hughes Medical Institute, is a leading authority on the structure and dynamics of microtubules. In this latest study, she and her group used cryo-EM, in which protein samples are flash-frozen at liquid nitrogen temperatures to preserve their natural structure, to determine microtubule structures in different nucleotide states with and without EB3. With cryo-EM and their image analysis methodology, they achieved a resolution of 3.5 Angstroms, a record for microtubules. For perspective, the diameter of a hydrogen atom is about 1.0 Angstroms.

    “We can now study the atomic details of microtubule polymerization and depolymerization to develop a complete description of microtubule dynamics,” Nogales says.

    Beyond their importance to our understanding of basic cell biology, microtubules are a major target for anticancer drugs, such as Taxol, which can prevent the transition from growing to shrinking nucleotide states or vice versa.

    Rui Zhang is the lead author of a Cell paper describing the record 3.5 Angstroms resolution imaging of microtubules

    “A better understanding of how microtubule dynamic instability is regulated could open new opportunities for improving the potency and selectivity of existing anti-cancer drugs, as well as facilitate the development of novel agents,” Nogales says.

    Nogales is the corresponding author of a paper describing this research in the journal Cell. The paper is entitled Mechanistic Origin of Microtubule Dynamic Instability and Its Modulation by EB Proteins. Co-authors are Rui Zhang, Gregory Alushin and Alan Brown.

    This work was funded by a grant from NIH’s National Institute of General Medical Sciences.

    See the full article here.

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  • richardmitnick 9:31 am on July 30, 2015 Permalink | Reply
    Tags: , Biology, , , ,   

    From livescience: “Origin-of-Life Story May Have Found Its Missing Link” 


    June 06, 2015
    Jesse Emspak

    A field of geysers called El Tatio located in northern Chile’s Andes Mountains. Credit: Gerald Prins

    How did life on Earth begin? It’s been one of modern biology’s greatest mysteries: How did the chemical soup that existed on the early Earth lead to the complex molecules needed to create living, breathing organisms? Now, researchers say they’ve found the missing link.

    Between 4.6 billion and 4.0 billion years ago, there was probably no life on Earth. The planet’s surface was at first molten and even as it cooled, it was getting pulverized by asteroids and comets. All that existed were simple chemicals. But about 3.8 billion years ago, the bombardment stopped, and life arose. Most scientists think the “last universal common ancestor” — the creature from which everything on the planet descends — appeared about 3.6 billion years ago.

    But exactly how that creature arose has long puzzled scientists. For instance, how did the chemistry of simple carbon-based molecules lead to the information storage of ribonucleic acid, or RNA?

    A hairpin loop from a pre-mRNA. Highlighted are the nucleobases (green) and the ribose-phosphate backbone (blue). Note that this is a single strand of RNA that folds back upon itself.

    The RNA molecule must store information to code for proteins. (Proteins in biology do more than build muscle — they also regulate a host of processes in the body.)

    The new research — which involves two studies, one led by Charles Carter and one led by Richard Wolfenden, both of the University of North Carolina — suggests a way for RNA to control the production of proteins by working with simple amino acids that does not require the more complex enzymes that exist today. [7 Theories on the Origin of Life on Earth]

    Missing RNA link

    This link would bridge this gap in knowledge between the primordial chemical soup and the complex molecules needed to build life. Current theories say life on Earth started in an “RNA world,” in which the RNA molecule guided the formation of life, only later taking a backseat to DNA, which could more efficiently achieve the same end result.

    The structure of the DNA double helix. The atoms in the structure are colour-coded by element and the detailed structure of two base pairs are shown in the bottom right.

    Like DNA, RNA is a helix-shaped molecule that can store or pass on information. (DNA is a double-stranded helix, whereas RNA is single-stranded.) Many scientists think the first RNA molecules existed in a primordial chemical soup — probably pools of water on the surface of Earth billions of years ago. [Photo Timeline: How the Earth Formed]

    The idea was that the very first RNA molecules formed from collections of three chemicals: a sugar (called a ribose); a phosphate group, which is a phosphorus atom connected to oxygen atoms; and a base, which is a ring-shaped molecule of carbon, nitrogen, oxygen and hydrogen atoms. RNA also needed nucleotides, made of phosphates and sugars.

    The question: How did the nucleotides come together within the soupy chemicals to make RNA? John Sutherland, a chemist at the University of Cambridge in England, published a study in May in the journal Nature Chemistry that showed that a cyanide-based chemistry could make two of the four nucleotides in RNA and many amino acids.

    That still left questions, though. There wasn’t a good mechanism for putting nucleotides together to make RNA. Nor did there seem to be a natural way for amino acids to string together and form proteins. Today, adenosine triphosphate (ATP) does the job of linking amino acids into proteins, activated by an enzyme called aminoacyl tRNA synthetase. But there’s no reason to assume there were any such chemicals around billions of years ago.

    Also, proteins have to be shaped a certain way in order to function properly. That means RNA has to be able to guide their formation — it has to “code” for them, like a computer running a program to do a task.

    Carter noted that it wasn’t until the past decade or two that scientists were able to duplicate the chemistry that makes RNA build proteins in the lab. “Basically, the only way to get RNA was to evolve humans first,” he said. “It doesn’t do it on its own.”

    Perfect sizes

    In one of the new studies, Carter looked at the way a molecule called “transfer RNA,” or tRNA, reacts with different amino acids.

    They found that one end of the tRNA could help sort amino acids according to their shape and size, while the other end could link up with amino acids of a certain polarity. In that way, this tRNA molecule could dictate how amino acids come together to make proteins, as well as determine the final protein shape. That’s similar to what the ATP enzyme does today, activating the process that strings together amino acids to form proteins.

    Carter told Live Science that the ability to discriminate according to size and shape makes a kind of “code” for proteins called peptides, which help to preserve the helix shape of RNA.

    “It’s an intermediate step in the development of genetic coding,” he said.

    In the other study, Wolfenden and colleagues tested the way proteins fold in response to temperature, since life somehow arose from a proverbial boiling pot of chemicals on early Earth. They looked at life’s building blocks, amino acids, and how they distribute in water and oil — a quality called hydrophobicity. They found that the amino acids’ relationships were consistent even at high temperatures — the shape, size and polarity of the amino acids are what mattered when they strung together to form proteins, which have particular structures.

    “What we’re asking here is, ‘Would the rules of folding have been different?'” Wolfenden said. At higher temperatures, some chemical relationships change because there is more thermal energy. But that wasn’t the case here.

    By showing that it’s possible for tRNA to discriminate between molecules, and that the links can work without “help,” Carter thinks he’s found a way for the information storage of chemical structures like tRNA to have arisen — a crucial piece of passing on genetic traits. Combined with the work on amino acids and temperature, it offers insight into how early life might have evolved.

    This work still doesn’t answer the ultimate question of how life began, but it does show a mechanism for the appearance of the genetic codes that pass on inherited traits, which got evolution rolling.

    The two studies are published in the June 1 issue of the journal Proceedings of the National Academy of Sciences.

    See the full article here.

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  • richardmitnick 4:04 pm on July 28, 2015 Permalink | Reply
    Tags: , Biology, , , ,   

    From BNL: “New Computer Model Could Explain how Simple Molecules Took First Step Toward Life” 

    Brookhaven Lab

    July 28, 2015
    Alasdair Wilkins

    Two Brookhaven researchers developed theoretical model to explain the origins of self-replicating molecules

    Brookhaven researchers Sergei Maslov (left) and Alexi Tkachenko developed a theoretical model to explain molecular self-replication.

    Nearly four billion years ago, the earliest precursors of life on Earth emerged. First small, simple molecules, or monomers, banded together to form larger, more complex molecules, or polymers. Then those polymers developed a mechanism that allowed them to self-replicate and pass their structure on to future generations.

    We wouldn’t be here today if molecules had not made that fateful transition to self-replication. Yet despite the fact that biochemists have spent decades searching for the specific chemical process that can explain how simple molecules could make this leap, we still don’t really understand how it happened.

    Now Sergei Maslov, a computational biologist at the U.S. Department of Energy’s Brookhaven National Laboratory and adjunct professor at Stony Brook University, and Alexei Tkachenko, a scientist at Brookhaven’s Center for Functional Nanomaterials (CFN), have taken a different, more conceptual approach. They’ve developed a model that explains how monomers could very rapidly make the jump to more complex polymers. And what their model points to could have intriguing implications for CFN’s work in engineering artificial self-assembly at the nanoscale. Their work is published in the July 28, 2015 issue of The Journal of Chemical Physics.

    To understand their work, let’s consider the most famous organic polymer, and the carrier of life’s genetic code: DNA. This polymer is composed of long chains of specific monomers called nucleotides, of which the four kinds are adenine, thymine, guanine, and cytosine (A, T, G, C). In a DNA double helix, each specific nucleotide pairs with another: A with T, and G with C. Because of this complementary pairing, it would be possible to put a complete piece of DNA back together even if just one of the two strands was intact.

    While DNA has become the molecule of choice for encoding biological information, its close cousin RNA likely played this role at the dawn of life. This is known as the RNA world hypothesis, and it’s the scenario that Maslov and Tkachenko considered in their work.

    The single complete RNA strand is called a template strand, and the use of a template to piece together monomer fragments is what is known as template-assisted ligation. This concept is at the crux of their work. They asked whether that piecing together of complementary monomer chains into more complex polymers could occur not as the healing of a broken polymer, but rather as the formation of something new.

    “Suppose we don’t have any polymers at all, and we start with just monomers in a test tube,” explained Tkachenko. “Will that mixture ever find its way to make those polymers? The answer is rather remarkable: Yes, it will! You would think there is some chicken-and-egg problem—that, in order to make polymers, you already need polymers there to provide the template for their formation. Turns out that you don’t really.”

    Instilling memory

    A schematic drawing of template-assisted ligation, shown in this model to give rise to autocatalytic systems. No image credit.

    Maslov and Tkachenko’s model imagines some kind of regular cycle in which conditions change in a predictable fashion—say, the transition between night and day. Imagine a world in which complex polymers break apart during the day, then repair themselves at night. The presence of a template strand means that the polymer reassembles itself precisely as it was the night before. That self-replication process means the polymer can transmit information about itself from one generation to the next. That ability to pass information along is a fundamental property of life.

    “The way our system replicates from one day cycle to the next is that it preserves a memory of what was there,” said Maslov. “It’s relatively easy to make lots of long polymers, but they will have no memory. The template provides the memory. Right now, we are solving the problem of how to get long polymer chains capable of memory transmission from one unit to another to select a small subset of polymers out of an astronomically large number of solutions.”

    According to Maslov and Tkachenko’s model, a molecular system only needs a very tiny percentage of more complex molecules—even just dimers, or pairs of identical molecules joined together—to start merging into the longer chains that will eventually become self-replicating polymers. This neatly sidesteps one of the most vexing puzzles of the origins of life: Self-replicating chains likely need to be very specific sequences of at least 100 paired monomers, yet the odds of 100 such pairs randomly assembling themselves in just the right order is practically zero.

    “If conditions are right, there is what we call a first-order transition, where you go from this soup of completely dispersed monomers to this new solution where you have these long chains appearing,” said Tkachenko. “And we now have this mechanism for the emergence of these polymers that can potentially carry information and transmit it downstream. Once this threshold is passed, we expect monomers to be able to form polymers, taking us from the primordial soup to a primordial soufflé.”

    While the model’s concept of template-assisted ligation does describe how DNA—as well as RNA—repairs itself, Maslov and Tkachenko’s work doesn’t require that either of those was the specific polymer for the origin of life.

    “Our model could also describe a proto-RNA molecule. It could be something completely different,” Maslov said.

    Order from disorder

    The fact that Maslov and Tkachenko’s model doesn’t require the presence of a specific molecule speaks to their more theoretical approach.

    “It’s a different mentality from what a biochemist would do,” said Tkachenko. “A biochemist would be fixated on specific molecules. We, being ignorant physicists, tried to work our way from a general conceptual point of view, as there’s a fundamental problem.”

    That fundamental problem is the second law of thermodynamics, which states that systems tend toward increasing disorder and lack of organization. The formation of long polymer chains from monomers is the precise opposite of that.

    “How do you start with the regular laws of physics and get to these laws of biology which makes things run backward, which make things more complex, rather than less complex?” Tkachenko queried. “That’s exactly the jump that we want to understand.”

    Applications in nanoscience

    The work is an outgrowth of efforts at the Center for Functional Nanomaterials, a DOE Office of Science User Facility, to use DNA and other biomolecules to direct the self-assembly of nanoparticles into large, ordered arrays. While CFN doesn’t typically focus on these kinds of primordial biological questions, Maslov and Tkachenko’s modeling work could help CFN scientists engaged in cutting-edge nanoscience research to engineer even larger and more complex assemblies using nanostructured building blocks.

    “There is a huge interest in making engineered self-assembled structures, so we were essentially thinking about two problems at once,” said Tkachenko. “One is relevant to biologists, and second asks whether we can engineer a nanosystem that will do what our model does.”

    The next step will be to determine whether template-aided ligation can allow polymers to begin undergoing the evolutionary changes that characterize life as we know it. While this first round of research involved relatively modest computational resources, that next phase will require far more involved models and simulations.

    Maslov and Tkachenko’s work has solved the problem of how long polymer chains capable of information transmission from one generation to the next could emerge from the world of simple monomers. Now they are turning their attention to how such a system could naturally narrow itself down from exponentially many polymers to only a select few with desirable sequences.

    “What we needed to show here was that this template-based ligation does result in a set of polymer chains, starting just from monomers,” said Tkachenko. “So the next question we will be asking is whether, because of this template-based merger, we will be able to see specific sequences that will be more ‘fit’ than others. So this work sets the stage for the shift to the Darwinian phase.”

    This work was supported by the DOE Office of Science.

    See the full article here.

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    One of ten national laboratories overseen and primarily funded by the Office of Science of the U.S. Department of Energy (DOE), Brookhaven National Laboratory conducts research in the physical, biomedical, and environmental sciences, as well as in energy technologies and national security. Brookhaven Lab also builds and operates major scientific facilities available to university, industry and government researchers. The Laboratory’s almost 3,000 scientists, engineers, and support staff are joined each year by more than 5,000 visiting researchers from around the world.Brookhaven is operated and managed for DOE’s Office of Science by Brookhaven Science Associates, a limited-liability company founded by Stony Brook University, the largest academic user of Laboratory facilities, and Battelle, a nonprofit, applied science and technology organization.

  • richardmitnick 10:40 am on May 8, 2015 Permalink | Reply
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    From AAAS: “Electron microscopes close to imaging individual atoms” 



    7 May 2015
    Robert F. Service

    This composite image of the protein β-galactosidase shows the progression of cryo-EM’s ability to resolve a protein’s features from mere blobs (left) a few years ago to the ultrafine 0.22-nanometer resolution today (right). Veronica Falconieri/ Subramaniam Lab/CCR/ NCI/ NIH

    Today’s digital photos are far more vivid than just a few years ago, thanks to a steady stream of advances in optics, detectors, and software. Similar advances have also improved the ability of machines called cryo-electron microscopes (cryo-EMs) to see the Lilliputian world of atoms and molecules. Now, researchers report that they’ve created the highest ever resolution cryo-EM image, revealing a druglike molecule bound to its protein target at near atomic resolution. The resolution is so sharp that it rivals images produced by x-ray crystallography, long the gold standard for mapping the atomic contours of proteins. This newfound success is likely to dramatically help drugmakers design novel medicines for a wide variety of conditions.

    “This represents a new era in imaging of proteins in humans with immense implications for drug design,” says Francis Collins, who heads the U.S. National Institutes of Health in Bethesda, Maryland. Collins may be partial. He’s the boss of the team of researchers from the National Cancer Institute (NCI) and the National Heart, Lung, and Blood Institute that carried out the work. Still, others agree that the new work represents an important milestone. “It’s a major advance in the technology,” says Wah Chiu, a cryo-EM structural biologist at Baylor College of Medicine in Houston, Texas. “It shows [cryo-EM] technology is here.”

    Cryo-EM has long seemed behind the times—an old hand tool compared with the modern power tools of structural biology. The two main power tools, x-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, enable researchers to pin down the position of protein features to less than 0.2 nanometers, good enough to see individual atoms. By contrast, cryo-EM has long been limited to a resolution of 0.5 nm or more.

    Cryo-EM works by firing a beam of electrons at a thin film containing myriad copies of a protein that have been instantly frozen in place by plunging them in liquid nitrogen. Detectors track the manner in which electrons scatter off different atoms in the protein. When an image is taken, the proteins are strewn about in random orientations. So researchers use imaging software to do two things; first, they align their images of individual proteins into a common orientation. Then, they use the electron scattering data to reconstruct the most likely position of all the protein’s amino acids and—if possible—its atoms.

    Cryo-EM has been around for decades. But until recently its resolution hasn’t even been close to crystallography and NMR. “We used to be called the field of blob-ology,” says Sriram Subramaniam, a cryo-EM structural biologist at NCI, who led the current project. But steady improvements to the electron beam generators, detectors, and imaging analysis software have slowly helped cryo-EM inch closer to the powerhouse techniques. Earlier this year, for example, two groups of researchers broke the 0.3-nm-resolution benchmark, enough to get a decent view of the side arms of two proteins’ individual amino acids. Still, plenty of detail in the images remained fuzzy.

    For their current study, Subramaniam and his colleagues sought to refine their images of β-galactosidase, a protein they imaged last year at a resolution of 0.33 nm. The protein serves as a good test case, Subramaniam says, because researchers can compare their images to existing x-ray structures to check their accuracy. Subramaniam adds that the current advance was more a product of painstaking refinements to a variety of techniques—including protein purification procedures that ensure each protein copy is identical and software improvements that allow researchers to better align their images. Subramaniam and his colleagues used some 40,000 separate images to piece together the final shape of their molecule. They report online today in Science that these refinements allowed them to produce a cryo-EM image of β-galactosidase at a resolution of 0.22 nm, not quite sharp enough to see individual atoms, but clear enough to see water molecules that bind to the protein in spots critical to the function of the molecule.

    That level of detail is equal to the resolution of many structures using x-ray crystallography, Chiu says. That’s vital, he adds, because for x-ray crystallography to work, researchers must produce millions of identical copies of a protein and then coax them to align in exactly the same orientation as they solidify into a crystal. But many proteins resist falling in line, making it impossible to determine their x-ray structure. NMR spectroscopy doesn’t require crystals, but it works only on small proteins. Cryo-EM represents the best of both worlds: It can work with massive proteins, but it doesn’t require crystals.

    As a result, the new advances could help structural biologists map vast numbers of new proteins they’ve never mapped before, Chiu says. That, in turn, could help drug developers design novel drugs for a multitude of conditions associated with different proteins. But one thing the technique has already shown is crystal clear, that in imaging, as well as biology, slow, evolutionary advances over time can produce big results.

    See the full article here.

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  • richardmitnick 11:37 am on March 24, 2015 Permalink | Reply
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    From Rockefeller: “Chemical tag marks future microRNAs for processing, study shows” 

    Rockefeller U bloc

    Rockefeller University

    March 24, 2015
    Zach Veilleux | 212-327-8982

    Molecular sorting machine: Scientists found the enzyme METTL3 (green) tags a particular sequence within RNA molecules destined to become gene-regulating microRNAs. While this happens within cells’ nuclei (blue), METTL3 is also found outside the nucleus in cells’ cytoplasm, as shown above.

    Just as two DNA strands naturally arrange themselves into a helix, DNA’s molecular cousin RNA can form hairpin-like loops. But unlike DNA, which has a single job, RNA can play many parts — including acting as a precursor for small molecules that block the activity of genes. These small RNA molecules must be trimmed from long hairpin-loop structures, raising a question: How do cells know which RNA loops need to be processed this way and which don’t?

    New research at Rockefeller University, published March 18 in Nature, reveals how cells sort out the loops meant to encode small RNAs, known as microRNAs, by tagging them with a chemical group. Because microRNAs help control processes throughout the body, this discovery has wide-ranging implications for development, health and disease, including cancer, the entry point for this research.

    “Work in our lab and elsewhere has shown changes in levels of microRNAs in a number of cancers. To better understand how and why this happens, we needed to first answer a more basic question and take a closer look at how cells normally identify and process microRNAs,” says study author Sohail Tavazoie, Leon Hess Associate Professor, Senior Attending Physician and head of the Elizabeth and Vincent Meyer Laboratory of Systems Cancer Biology. “Claudio Alarcón, a research associate in my lab, has discovered that cells can increase or decrease microRNAs by using a specific chemical tag.”

    Long known as the intermediary between DNA and proteins, RNA has turned out to be a versatile molecule. Scientists have discovered a number of RNA molecules, including microRNAs that regulate gene expression. MicroRNAs are encoded into the genome as DNA, then transcribed into hairpin loop RNA molecules, known as primary microRNAs. These loops are then clipped to generate microRNA precursors.

    To figure out how cells know which hairpin loops to start trimming, Alarcón set out to look for modifications cells might make to the RNA molecules that are destined to become microRNAs. Using bioinformatics software, he scanned for unusual patterns in the unprocessed RNA sequences. The sequence GGAC, code for the bases guanine-guanine-adenine-cytosine, stood out because it appeared with surprising frequency in the unprocessed primary microRNAs. GGAC, in turn, led the researchers to an enzyme known as METTL3, which tags the GGAC segments with a chemical marker, a methyl group, at a particular spot on the adenine.

    “Once we arrived at METTL3, everything made sense. The methyl in adenosines (m6A tag) is the most common known RNA modification. METTL3 is known to contribute to stabilizing and processing messenger RNA, which is transcribed from DNA, but it is suspected of doing much more,” Alarcón says. “Now, we have evidence for a third role: the processing of primary microRNAs.”

    In series of experiments, the researchers confirmed the importance of methyl tagging, finding high levels of it near all types of unprocessed microRNAs, suggesting it is a generic mark associated with these molecules. When they reduced expression of METTL3, unprocessed primary microRNAs accumulated, indicating that the enzyme’s tagging action was important to the process. And, working in cell culture and in biochemical systems, they found primary microRNAs were processed much more efficiently in the presence of the methyl tags than without them.

    “Cells can remove these tags, as well as add them, so these experiments have identified a switch that can be used to ramp up or tamp down microRNA levels, and as a result, alter gene expression,” Tavazoie says. “Not only do we see abnormalities in microRNAs in cancer, levels of METTL3 can be altered as well, which suggests this pathway is could govern cancer progression.”

    See the full article here.

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    The Rockefeller University is a world-renowned center for research and graduate education in the biomedical sciences, chemistry, bioinformatics and physics. The university’s 76 laboratories conduct both clinical and basic research and study a diverse range of biological and biomedical problems with the mission of improving the understanding of life for the benefit of humanity.

    Founded in 1901 by John D. Rockefeller, the Rockefeller Institute for Medical Research was the country’s first institution devoted exclusively to biomedical research. The Rockefeller University Hospital was founded in 1910 as the first hospital devoted exclusively to clinical research. In the 1950s, the institute expanded its mission to include graduate education and began training new generations of scientists to become research leaders around the world. In 1965, it was renamed The Rockefeller University.

  • richardmitnick 4:01 am on March 21, 2015 Permalink | Reply
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    From NOVA: “Genetically Engineering Almost Anything” 2014 and Very Important 



    17 Jul 2014
    Tim De Chant and Eleanor Nelsen

    When it comes to genetic engineering, we’re amateurs. Sure, we’ve known about DNA’s structure for more than 60 years, we first sequenced every A, T, C, and G in our bodies more than a decade ago, and we’re becoming increasingly adept at modifying the genes of a growing number of organisms.

    But compared with what’s coming next, all that will seem like child’s play. A new technology just announced today has the potential to wipe out diseases, turn back evolutionary clocks, and reengineer entire ecosystems, for better or worse. Because of how deeply this could affect us all, the scientists behind it want to start a discussion now, before all the pieces come together over the next few months or years. This is a scientific discovery being played out in real time.

    Scientists have figured out how to use a cell’s DNA repair mechanisms to spread traits throughout a population.

    Today, researchers aren’t just dropping in new genes, they’re deftly adding, subtracting, and rewriting them using a series of tools that have become ever more versatile and easier to use. In the last few years, our ability to edit genomes has improved at a shockingly rapid clip. So rapid, in fact, that one of the easiest and most popular tools, known as CRISPR-Cas9, is just two years old. Researchers once spent months, even years, attempting to rewrite an organism’s DNA. Now they spend days.

    Soon, though, scientists will begin combining gene editing with gene drives, so-called selfish genes that appear more frequently in offspring than normal genes, which have about a 50-50 chance of being passed on. With gene drives—so named because they drive a gene through a population—researchers just have to slip a new gene into a drive system and let nature take care of the rest. Subsequent generations of whatever species we choose to modify—frogs, weeds, mosquitoes—will have more and more individuals with that gene until, eventually, it’s everywhere.

    Cas9-based gene drives could be one of the most powerful technologies ever discovered by humankind. “This is one of the most exciting confluences of different theoretical approaches in science I’ve ever seen,” says Arthur Caplan, a bioethicist at New York University. “It merges population genetics, genetic engineering, molecular genetics, into an unbelievably powerful tool.”

    We’re not there yet, but we’re extraordinarily close. “Essentially, we have done all of the pieces, sometimes in the same relevant species.” says Kevin Esvelt, a postdoc at Harvard University and the wunderkind behind the new technology. “It’s just no one has put it all together.”

    It’s only a matter of time, though. The field is progressing rapidly. “We could easily have laboratory tests within the next few months and then field tests not long after that,” says George Church, a professor at Harvard University and Esvelt’s advisor. “That’s if everybody thinks it’s a good idea.”

    It’s likely not everyone will think this is a good idea. “There are clearly people who will object,” Caplan says. “I think the technique will be incredibly controversial.” Which is why Esvelt, Church, and their collaborators are publishing papers now, before the different parts of the puzzle have been assembled into a working whole.

    “If we’re going to talk about it at all in advance, rather than in the past tense,” Church says, “now is the time.”

    “Deleterious Genes”

    The first organism Esvelt wants to modify is the malaria-carrying mosquito Anopheles gambiae. While his approach is novel, the idea of controlling mosquito populations through genetic modification has actually been around since the late 1970s. Then, Edward F. Knipling, an entomologist with the U.S. Department of Agriculture, published a substantial handbook with a chapter titled “Use of Insects for Their Own Destruction.” One technique, he wrote, would be to modify certain individuals to carry “deleterious genes” that could be passed on generation after generation until they pervaded the entire population. It was an idea before its time. Knipling was on the right track, but he and his contemporaries lacked the tools to see it through.

    The concept surfaced a few more times before being picked up by Austin Burt, an evolutionary biologist and population geneticist at Imperial College London. It was the late 1990s, and Burt was busy with his yeast cells, studying their so-called homing endonucleases, enzymes that facilitate the copying of genes that code for themselves. Self-perpetuating genes, if you will. “Through those studies, gradually, I became more and more familiar with endonucleases, and I came across the idea that you might be able to change them to recognize new sequences,” Burt recalls.

    Other scientists were investigating endonucleases, too, but not in the way Burt was. “The people who were thinking along those lines, molecular biologists, were thinking about using these things for gene therapy,” Burt says. “My background in population biology led me to think about how they could be used to control populations that were particularly harmful.”

    In 2003, Burt penned an influential article that set the course for an entire field: We should be using homing endonucleases, a type of gene drive, to modify malaria-carrying mosquitoes, he said, not ourselves. Burt saw two ways of going about it—one, modify a mosquito’s genome to make it less hospitable to malaria, and two, skew the sex ratio of mosquito populations so there are no females for the males to reproduce with. In the following years, Burt and his collaborators tested both in the lab and with computer models before they settled on sex ratio distortion. (Making mosquitoes less hospitable to malaria would likely be a stopgap measure at best; the Plasmodium protozoans could evolve to cope with the genetic changes, just like they have evolved resistance to drugs.)

    Burt has spent the last 11 years refining various endonucleases, playing with different scenarios of inheritance, and surveying people in malaria-infested regions. Now, he finally feels like he is closing in on his ultimate goal. “There’s a lot to be done still,” he says. “But on the scale of years, not months or decades.”

    Cheating Natural Selection

    Cas9-based gene drives could compress that timeline even further. One half of the equation—gene drives—are the literal driving force behind proposed population-scale genetic engineering projects. They essentially let us exploit evolution to force a desired gene into every individual of a species. “To anthropomorphize horribly, the goal of a gene is to spread itself as much as possible,” Esvelt says. “And in order to do that, it wants to cheat inheritance as thoroughly as it can.” Gene drives are that cheat.

    Without gene drives, traits in genetically-engineered organisms released into the wild are vulnerable to dilution through natural selection. For organisms that have two parents and two sets of chromosomes (which includes humans, many plants, and most animals), traits typically have only a 50-50 chance of being inherited, give or take a few percent. Genes inserted by humans face those odds when it comes time to being passed on. But when it comes to survival in the wild, a genetically modified organism’s odds are often less than 50-50. Engineered traits may be beneficial to humans, but ultimately they tend to be detrimental to the organism without human assistance. Even some of the most painstakingly engineered transgenes will be gradually but inexorably eroded by natural selection.

    Some naturally occurring genes, though, have over millions of years learned how to cheat the system, inflating their odds of being inherited. Burt’s “selfish” endonucleases are one example. They take advantage of the cell’s own repair machinery to ensure that they show up on both chromosomes in a pair, giving them better than 50-50 odds when it comes time to reproduce.

    A gene drive (blue) always ends up in all offspring, even if only one parent has it. That means that, given enough generations, it will eventually spread through the entire population.

    Here’s how it generally works. The term “gene drive” is fairly generic, describing a number of different systems, but one example involves genes that code for an endonuclease—an enzyme which acts like a pair of molecular scissors—sitting in the middle of a longer sequence of DNA that the endonculease is programmed to recognize. If one chromosome in a pair contains a gene drive but the other doesn’t, the endonuclease cuts the second chromosome’s DNA where the endonuclease code appears in the first.

    The broken strands of DNA trigger the cell’s repair mechanisms. In certain species and circumstances, the cell unwittingly uses the first chromosome as a template to repair the second. The repair machinery, seeing the loose ends that bookend the gene drive sequence, thinks the middle part—the code for the endonuclease—is missing and copies it onto the broken chromosome. Now both chromosomes have the complete gene drive. The next time the cell divides, splitting its chromosomes between the two new cells, both new cells will end up with a copy of the gene drive, too. If the entire process works properly, the gene drive’s odds of inheritance aren’t 50%, but 100%.

    Here, a mosquito with a gene drive (blue) mates with a mosquito without one (grey). In the offspring, one chromosome will have the drive. The endonuclease then slices into the drive-free DNA. When the strand gets repaired, the cell’s machinery uses the drive chromosome as a template, unwittingly copying the drive into the break.

    Most natural gene drives are picky about where on a strand of DNA they’ll cut, so they need to be modified if they’re to be useful for genetic engineering. For the last few years, geneticists have tried using genome-editing tools to build custom gene drives, but the process was laborious and expensive. With the discovery of CRISPR-Cas9 as a genome editing tool in 2012, though, that barrier evaporated. CRISPR is an ancient bacterial immune system which identifies the DNA of invading viruses and sends in an endonuclease, like Cas9, to chew it up. Researchers quickly realized that Cas9 could easily be reprogrammed to recognize nearly any sequence of DNA. All that’s needed is the right RNA sequence—easily ordered and shipped overnight—which Cas9 uses to search a strand of DNA for where to cut. This flexibility, Esvelt says, “lets us target, and therefore edit, pretty much anything we want.” And quickly.

    Gene drives and Cas9 are each powerful on their own, but together they could significantly change biology. CRISRP-Cas9 allows researchers to edit genomes with unprecedented speed, and gene drives allow engineered genes to cheat the system, even if the altered gene weakens the organism. Simply by being coupled to a gene drive, an engineered gene can race throughout a population before it is weeded out. “Eventually, natural selection will win,” Esvelt says, but “gene drives just let us get ahead of the game.”

    Beyond Mosquitoes

    If there’s anywhere we could use a jump start, it’s in the fight against malaria. Each year, the disease kills over 200,000 people and sickens over 200 million more, most of whom are in Africa. The best new drugs we have to fight it are losing ground; the Plasmodium parasite is evolving resistance too quickly.

    False-colored electron micrograph of a Plasmodium sp. sporozoite.

    And we’re nowhere close to releasing an effective vaccine. The direct costs of treating the disease are estimated at $12 billion, and the economies of affected countries grew 1.3% less per year, a substantial amount.

    Which is why Esvelt and Burt are both so intently focused on the disease. “If we target the mosquito, we don’t have to face resistance on the parasite itself. The idea is, we can just take out the vector and stop all transmission. It might even lead to eradication,” Esvelt says.

    Esvelt initially mulled over the idea of building Cas9-based gene drives in mosquitoes to do just that. He took the idea to to Flaminia Catteruccia, a professor who studies malaria at the Harvard School of Public Health, and the two grew increasingly certain that such a system would not only work, but work well. As their discussions progressed, though, Esvelt realized they were “missing the forest for the trees.” Controlling malaria-carrying mosquitoes was just the start. Cas9-based gene drives were the real breakthrough. “If it let’s us do this for mosquitos, what is to stop us from potentially doing it for almost anything that is sexually reproducing?” he realized.

    In theory, nothing. But in reality, the system works best on fast-reproducing species, Esvelt says. Short generation times allow the trait to spread throughout a population more quickly. Mosquitoes are a perfect test case. If everything were to work perfectly, deleterious traits could sweep through populations of malaria-carrying mosquitoes in as few as five years, wiping them off the map.

    Other noxious species could be candidates, too. Certain invasive species, like mosquitoes in Hawaii or Asian carp in the Great Lakes, could be targeted with Cas9-based gene drives to either reduce their numbers or eliminate them completely. Agricultural weeds like horseweed that have evolved resistance to glyphosate, a herbicide that is broken down quickly in the soil, could have their susceptibility to the compound reintroduced, enabling more farmers to adopt no-till practices, which help conserve topsoil. And in the more distant future, Esvelt says, weeds could even be engineered to introduce vulnerabilities to completely benign substances, eliminating the need for toxic pesticides. The possibilities seem endless.

    The Decision

    Before any of that can happen, though, Esvelt and Church are adamant that the public help decide whether the research should move forward. “What we have here is potentially a general tool for altering wild populations,” Esvelt says. “We really want to make sure that we proceed down this path—if we decide to proceed down this path—as safely and responsibly as possible.”

    To kickstart the conversation, they partnered with the MIT political scientist Kenneth Oye and others to convene a series of workshops on the technology. “I thought it might be useful to get into the room people with slightly different material interests,” Oye says, so they invited regulators, nonprofits, companies, and environmental groups. The idea, he says, was to get people to meet several times, to gain trust and before “decisions harden.” Despite the diverse viewpoints, Oye says there was surprising agreement among participants about what the important outstanding questions were.

    As the discussion enters the public sphere, tensions are certain to intensify. “I don’t care if it’s a weed or a blight, people still are going to say this is way too massive a genetic engineering project,” Caplan says. “Secondly, it’s altering things that are inherited, and that’s always been a bright line for genetic engineering.” Safety, too, will undoubtedly be a concern. As the power of a tool increases, so does its potential for catastrophe, and Cas9-based gene drives could be extraordinarily powerful.

    There’s also little in the way of precedent that we can use as a guide. Our experience with genetically modified foods would seem to be a good place to start, but they are relatively niche organisms that are heavily dependent on water and fertilizer. It’s pretty easy to keep them contained to a field. Not so with wild organisms; their potential to spread isn’t as limited.

    Aware of this, Esvelt and his colleagues are proposing a number of safeguards, including reversal drives that can undo earlier engineered genes. “We need to really make sure those work if we’re proposing to build a drive that is intended to modify a wild population,” Esvelt says.

    There are still other possible hurdles to surmount—lab-grown mosquitoes may not interbreed with wild ones, for example—but given how close this technology is to prime time, Caplan suggests researchers hew to a few initial ethical guidelines. One, use species that are detrimental to human health and don’t appear to fill a unique niche in the wild. (Malaria-carrying mosquitoes seem fit that description.) Two, do as much work as possible using computer models. And three, researchers should continue to be transparent about their progress, as they have been. “I think the whole thing is hugely exciting,” Caplan says. “But the time to really get cracking on the legal/ethical infrastructure for this technology is right now.”

    Church agrees, though he’s also optimistic about the potential for Cas9-based gene drives. “I think we need to be cautious with all new technologies, especially all new technologies that are messing with nature in some way or another. But there’s also a risk of doing nothing,” Church says. “We have a population of 7 billion people. You have to deal with the environmental consequences of that.”

    See the full article here.

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  • richardmitnick 2:36 am on March 21, 2015 Permalink | Reply
    Tags: , Biology,   

    From Princeton: “Letting go of the (genetic) apron strings (Cell)” 

    Princeton University
    Princeton University

    March 20, 2015
    Catherine Zandonella, Office of the Dean for Research

    Cells in an early-stage fruit fly embryo. (Image courtesy of NIGMS image gallery).

    A new study from Princeton University researchers sheds light on the handing over of genetic control from mother to offspring early in development. Learning how organisms manage this transition could help researchers understand larger questions about how embryos regulate cell division and differentiation into new types of cells.

    The study, published in the March 12 issue of the journal Cell, provides new insight into the mechanism for this genetic hand-off, which happens within hours of fertilization, when the newly fertilized egg is called a zygote.

    “At the beginning, everything the embryo needs to survive is provided by mom, but eventually that stuff runs out, and the embryo needs to start making its own proteins and cellular machinery,” said Princeton postdoctoral researcher in the Department of Molecular Biology and first author Shelby Blythe. “We wanted to find out what controls that transition.”

    Blythe conducted the study with senior author Eric Wieschaus, Princeton’s Squibb Professor in Molecular Biology, Professor of Molecular Biology and the Lewis-Sigler Institute for Integrative Genomics, a Howard Hughes Medical Institute investigator, and a Nobel laureate in physiology or medicine.

    Researchers have known that in most animals, a newly fertilized egg cell divides rapidly, producing exact copies of itself using gene products supplied by the mother. After a short while, this rapid cell division pauses, and when it restarts, the embryonic DNA takes control and the cells divide much more slowly, differentiating into new cell types that are needed for the body’s organs and systems.

    To find out what controls this maternal to zygotic transition, also called the midblastula transition (MBT), Blythe conducted experiments in the fruit fly Drosophila melanogaster, which has long served as a model for development in higher organisms including humans.

    These experiments revealed that the slower cell division is a consequence of an upswing in DNA errors after the embryo’s genes take over. Cell division slows down because the cell’s DNA-copying machinery has to stop and wait until the damage is repaired.

    Blythe found that it wasn’t the overall amount of embryonic DNA that caused this increase in errors. Instead, his experiments indicated that the high error rate was due to molecules that bind to DNA to activate the reading, or “transcription,” of the genes. These molecules stick to the DNA strands at thousands of sites and prevent the DNA copying machinery from working properly.

    To discover this link between DNA errors and slower cell replication, Blythe used genetic techniques to create Drosophila embryos that were unable to repair DNA damage and typically died shortly after beginning to use their own genes. He then blocked the molecules that initiate the process of transcription of the zygotic genes, and found that the embryos survived, indicating that these molecules that bind to the DNA strands, called transcription factors, were triggering the DNA damage. He also discovered that a protein involved in responding to DNA damage, called Replication Protein A (RPA), appeared near the locations where DNA transcription was being initiated. “This provided evidence that the process of awakening the embryo’s genome is deleterious for DNA replication,” he said.

    The study also demonstrates a mechanism by which the developing embryo ensures that cell division happens at a pace that is slow enough to allow the repair of damage to DNA during the switchover from maternal to zygotic gene expression. “For the first time we have a mechanistic foothold on how this process works,” Blythe said.

    The work also enables researchers to explore larger questions of how embryos regulate DNA replication and transcription. “This study allows us to think about the idea that the ‘character’ of the DNA before and after the MBT has something to do with the DNA acquiring the architectural features of chromatin [the mix of DNA and proteins that make up chromosomes] that allow us to point to a spot and say ‘this is a gene’ and ‘this is not a gene’,” Blythe said. “Many of these features are indeed absent early in embryogenesis, and we suspect that the absence of these features is what allows the rapid copying of the DNA template early on. Part of what is so exciting about this is that early embryos may represent one of the only times when this chromatin architecture is missing or ‘blank’. Additionally, these early embryos allow us to study how the cell builds and installs these features that are so essential to the fundamental processes of cell biology.”

    This work was supported in part by grant 5R37HD15587 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development.

    Read the abstract.

    Blythe, Shelby A. & Eric R. Wieschaus. Zygotic Genome Activation Triggers the DNA Replication Checkpoint at the Midblastula Transition. Cell. Published online on March 5, 2015. doi:10.1016/j.cell.2015.01.050. http://www.sciencedirect.com/science/article/pii/S0092867415001282

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  • richardmitnick 2:23 am on March 21, 2015 Permalink | Reply
    Tags: , Biology,   

    From Carnegie: “Food-delivery process inside seeds revealed” 

    Carnegie Institution of Washington bloc

    Carnegie Institution of Washington

    Friday, March 20, 2015
    No Writer Credit

    A comparison of normal seeds and seeds lacking SWEETS 11, 12, and 15, which are wrinkled (similar to those [Gregor] Mendel used to track down the basic rules of genetics). Embryonic development is clearly retarded in these mutants because they are unable to move sugars from the seed’s coat to the embryo inside.

    Inside every seed is the embryo of a plant, and in most cases also a storage of food needed to power initial growth of the young seedling. A seed consists mainly of carbohydrates and these have to be is transported from the leaf where they are assimilated into the seed’s outer coat from the parent plant and then accessed by the embryo. If not enough food is delivered, the seeds won’t have the energy to grow when it’s time to germinate. But very little is understood about this delivery process.

    New work from a team led by Carnegie’s Wolf Frommer identifies biochemical pathways necessary for stocking the seed’s food supplies. These findings could be targeted when engineering crops for higher yields.

    Published in The Plant Cell, the research identifies three members of the SWEET family of sugar-transport proteins that are used to deliver the sugars that are produced in the plant’s leaves to the embryonic plant inside of a seed.

    Frommer’s lab has done extensive work on SWEET proteins, which have an array of functions in plants including nectar secretion. SWEET transporters are also vulnerable to takeover by pathogens, which thereby hijack the plant’s food and energy supplies.

    The research team—Carnegie’s Li-Quing Chen, I Winnie Lin, Xiao-Qing Qu, Davide Sosso, and Alejandra Loñdono, as well as Heather McFarlane and A. Lacey Samuels from the University of British Columbia—found that SWEETS 11, 12, and 15 funnel sucrose toward the developing plant embryos through multiple pathways.

    Specially created mutants that eliminate these three SWEET transporters show wrinkled seeds similar to those Mendel used to track down the basic rules of genetics. Embryonic development is clearly retarded in these mutants because they are unable to move sugars from the seed’s coat to the embryo inside.

    “Our findings answer long-held questions about embryonic plant nutrition and have major potential importance for improving crop yields,” Frommer said.

    See the full article here.

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    Andrew Carnegie established a unique organization dedicated to scientific discovery “to encourage, in the broadest and most liberal manner, investigation, research, and discovery and the application of knowledge to the improvement of mankind…” The philosophy was and is to devote the institution’s resources to “exceptional” individuals so that they can explore the most intriguing scientific questions in an atmosphere of complete freedom. Carnegie and his trustees realized that flexibility and freedom were essential to the institution’s success and that tradition is the foundation of the institution today as it supports research in the Earth, space, and life sciences.

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