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  • richardmitnick 2:33 pm on September 25, 2015 Permalink | Reply
    Tags: , , , Genetics,   

    From MIT: “New system for human genome editing has potential to increase power and precision of DNA engineering” 

    MIT News

    September 25, 2015
    Broad Institute

    CRISPR systems are found in many different bacterial species, and have evolved to protect host cells against infection by viruses. Image courtesy of Broad Institute/Science Photo Images

    CRISPR-Cpf1 offers simpler approach to editing DNA; technology could disrupt scientific and commercial landscape.

    A team including the scientist who first harnessed the CRISPR-Cas9 system for mammalian genome editing has now identified a different CRISPR system with the potential for even simpler and more precise genome engineering.

    In a study published today in Cell, Feng Zhang and his colleagues at the Broad Institute of MIT and Harvard and the McGovern Institute for Brain Research at MIT, with co-authors Eugene Koonin at the National Institutes of Health, Aviv Regev of the Broad Institute and the MIT Department of Biology, and John van der Oost at Wageningen University, describe the unexpected biological features of this new system and demonstrate that it can be engineered to edit the genomes of human cells.

    “This has dramatic potential to advance genetic engineering,” says Eric Lander, director of the Broad Institute. “The paper not only reveals the function of a previously uncharacterized CRISPR system, but also shows that Cpf1 can be harnessed for human genome editing and has remarkable and powerful features. The Cpf1 system represents a new generation of genome editing technology.”

    CRISPR sequences were first described in 1987, and their natural biological function was initially described in 2010 and 2011. The application of the CRISPR-Cas9 system for mammalian genome editing was first reported in 2013, by Zhang and separately by George Church at Harvard University.

    In the new study, Zhang and his collaborators searched through hundreds of CRISPR systems in different types of bacteria, searching for enzymes with useful properties that could be engineered for use in human cells. Two promising candidates were the Cpf1 enzymes from bacterial species Acidaminococcus and Lachnospiraceae, which Zhang and his colleagues then showed can target genomic loci in human cells.

    “We were thrilled to discover completely different CRISPR enzymes that can be harnessed for advancing research and human health,” says Zhang, the W.M. Keck Assistant Professor in Biomedical Engineering in MIT’s Department of Brain and Cognitive Sciences.

    The newly described Cpf1 system differs in several important ways from the previously described Cas9, with significant implications for research and therapeutics, as well as for business and intellectual property:

    First: In its natural form, the DNA-cutting enzyme Cas9 forms a complex with two small RNAs, both of which are required for the cutting activity. The Cpf1 system is simpler in that it requires only a single RNA. The Cpf1 enzyme is also smaller than the standard SpCas9, making it easier to deliver into cells and tissues.

    Second, and perhaps most significantly: Cpf1 cuts DNA in a different manner than Cas9. When the Cas9 complex cuts DNA, it cuts both strands at the same place, leaving “blunt ends” that often undergo mutations as they are rejoined. With the Cpf1 complex the cuts in the two strands are offset, leaving short overhangs on the exposed ends. This is expected to help with precise insertion, allowing researchers to integrate a piece of DNA more efficiently and accurately.

    Third: Cpf1 cuts far away from the recognition site, meaning that even if the targeted gene becomes mutated at the cut site, it can likely still be recut, allowing multiple opportunities for correct editing to occur.

    Fourth: The Cpf1 system provides new flexibility in choosing target sites. Like Cas9, the Cpf1 complex must first attach to a short sequence known as a PAM, and targets must be chosen that are adjacent to naturally occurring PAM sequences. The Cpf1 complex recognizes very different PAM sequences from those of Cas9. This could be an advantage in targeting some genomes, such as in the malaria parasite as well as in humans.

    “The unexpected properties of Cpf1 and more precise editing open the door to all sorts of applications, including in cancer research,” says Levi Garraway, an institute member of the Broad Institute, and the inaugural director of the Joint Center for Cancer Precision Medicine at the Dana-Farber Cancer Institute, Brigham and Women’s Hospital, and the Broad Institute. Garraway was not involved in the research.

    An open approach to empower research

    Zhang, along with the Broad Institute and MIT, plan to share the Cpf1 system widely. As with earlier Cas9 tools, these groups will make this technology freely available for academic research via the Zhang lab’s page on the plasmid-sharing website Addgene, through which the Zhang lab has already shared Cas9 reagents more than 23,000 times with researchers worldwide to accelerate research. The Zhang lab also offers free online tools and resources for researchers through its website.

    The Broad Institute and MIT plan to offer nonexclusive licenses to enable commercial tool and service providers to add this enzyme to their CRISPR pipeline and services, further ensuring availability of this new enzyme to empower research. These groups plan to offer licenses that best support rapid and safe development for appropriate and important therapeutic uses.

    “We are committed to making the CRISPR-Cpf1 technology widely accessible,” Zhang says. “Our goal is to develop tools that can accelerate research and eventually lead to new therapeutic applications. We see much more to come, even beyond Cpf1 and Cas9, with other enzymes that may be repurposed for further genome editing advances.”

    See the full article here .

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  • richardmitnick 12:13 pm on September 18, 2015 Permalink | Reply
    Tags: , Gene splicing, Genetics,   

    From New Scientist: “Gene editing: Bring it on” 


    New Scientist

    18 September 2015
    Jessica Griggs

    “Einstein said our technology exceeds our humanity. I don’t buy that” (Image: Jason Grow Photography)

    Breakthroughs in DNA technology are opening the door to a superhuman future. Genetic engineering pioneer George Church says we have nothing to fear.

    There has been a lot of excitement lately over the new gene-editing technology, CRISPR. How does it work?

    Gene editing is snipping out a targeted DNA sequence and replacing it with another. It used to be time-consuming and imprecise, but now you can edit any living genome, using your computer to target a stretch of DNA. Guides made of bespoke RNA lead the CRISPR molecular machinery to the target, where an enzyme makes a cut. This either destroys the function of the DNA in that location or allows you to change its functioning by manipulating how the cells repair the cut, for example by inserting a genetic sequence of your choice.

    What will we be able to do with CRISPR?

    It could enable gene therapies that would allow physicians to fix genetic diseases, including some types of blindness, the blood disorder beta thalassemia and the neurodegenerative disorder Tay-Sachs disease. It could also mean new approaches to treating cancers and viral infections, including HIV.

    Other techniques could allow helpful DNA to spread through wild animal populations – which may allow us to eliminate infectious diseases like malaria.

    Earlier this year, there was controversy when a team in China attempted to use CRISPR to edit the genes of non-viable human embryos. For some people this still crossed an ethical line. What are your thoughts?

    In the early days of any field, if you mess up enough, you don’t just mess up your lab, you mess up all the labs. I don’t think this brouhaha will kill gene editing – they didn’t hurt any patients. But they were incautious.

    From a technical point of view, there are several widely known techniques that have been proven to improve the specificity and efficacy of CRISPR gene editing, some of which were developed in my lab. The Chinese group must have known that their work was going to get a lot of attention, so it was disappointing that they chose not to use these techniques. They may have felt that if they waited to do it the right way, another lab would have scooped them. In practice these other labs, mine included, are not doing CRISPR research with human embryos, so I don’t know what they were worried about.

    Mediocre science is not the same as evil science – after all, the experiments they did are legal in most countries.

    So you’re not worried about the ethical issues that have been raised?

    For me, the safety issues are the ethical issues, and the safety issues are not fundamentally different from those of any new therapeutic. Gene-editing techniques are being tested in animals extensively, in primates as well as rodents, and will eventually move into people.

    I’m a professional worrier. But the form that my worry takes is action. As soon as somebody expresses something to me that they think is wrong with the world or with research, I say “let’s work on this”. We can innovate on safety measures.

    Some people are against any tinkering with the gene pool – summoning the ever-present spectre of designer babies.

    We humans already tinker with the gene pool with inherited diseases such as Tay-Sachs: genetic counselling before or after conception can help parents to decide on the health of their future children.

    We tinker with the gene pool every time we fly at high altitude, which increases random mutations in developing sperm and egg cells. These things are allowed. If you don’t like the concept of tinkering with the gene pool, then ban it across the board. Don’t be exceptional about CRISPR.

    “People worry that gene editing is irreversible, but that notion is a straw man”

    Then there’s the issue of what we value. Do we value long life, intelligence, athleticism, beauty? We are already making our children more educated than their ancestors, and typically we do that without the permission of the children. People pay for products that improve beauty and athleticism. If you don’t think those are good values, then ban tinkering with them. But be fair. Don’t ban a particular form of it – unless it’s unsafe.

    Enzymes (pale blue) cuts target DNA (red), guided by RNA (yellow) – it’s CRISPR in action (Image: Bang Wong, from source material provided by Feng Zhang)

    Should unborn children have the right not to have their parents genetically dictate who they become, or is it the parents’ choice?

    Parents owe it to their children to provide them with a good start. Education, chores, nutrition, dress code, faith and curfews are often dictated by parents for the good of the child. And parents already use genetic counselling to guide pre-conception and prenatal choices, to dictate that the child will not be burdened by serious genetic disease. Once those children grow up, they can choose different dictates for their own children as new information about genetics and child rearing becomes available.

    There are also concerns that genetic modification affecting sperm and eggs will produce changes that are irreversible and that can be passed down through the generations.

    The irreversibility argument strikes me as a straw man because, clearly, gene editing is not irreversible on a population-wide level.

    If the population is changing because individuals are making decisions, it can change back if governments or individuals make alternative decisions. In fact, it can change back more readily because the technology is improving all the time and the costs are reducing. Now, it could be that people won’t want to reverse the changes, but that’s telling you that the change is valuable in some way.

    If humanity doesn’t take the opportunity to advance genetic engineering in people, are we doing ourselves a disservice?

    Absolutely. This question should come up more frequently. With seven billion people and growing, sitting still is not really a great option. For example, we could wipe out malaria using a gene drive – a technique that would allow a malaria-resistance gene to spread through a population of mosquitoes extremely rapidly.

    That could be risky.

    Some of us may say that gene drives are too risky in general because of unknown unknowns – like perhaps causing the extinction of a mosquito species. But if you did, it’s unlikely that that is going to kill any other animal. And every year that we hesitate, 600,000 people die of malaria unnecessarily and another couple of million get sick and miss days at work. That’s a pretty big price to pay.

    You’re also interested in what’s called genetic augmentation. Isn’t using gene therapy in that way, whether for physical or mental enhancement, controversial too?

    It’s less controversial because it would be in consenting adults. If a majority agrees that adults can be augmented – using a new drug, device, or gene therapy – then as long as it passes the usual safety and efficacy rules of the US Food and Drug Administration (FDA), in my view it’s not controversial enough to prevent rapid adoption.

    You’ve famously come up with a list of 10 particularly protective but rare gene variants that we might all benefit from.

    Yes. For example, PCSK9 protects you from cardiovascular disease, and it’s “superhuman” in the sense that people who have it are well beyond the average human in terms of low cardiovascular risk. Three others on the list – MSTN, LRP, APOE – will probably lead to therapies to prevent muscle degeneration, osteoporosis and dementia.

    What about genes unrelated to disease? If we go in for editing such genes, what kind of society will we end up with?

    It comes down to what societies value, and then what individuals want.

    I don’t think everyone will want the same thing: we won’t all suddenly become exactly 5 feet 10 inches tall, with blonde hair and blue eyes. I think augmentation will actually increase diversity.

    Some of it will be driven by need or ambition. People who want to go to space may want super-strong bones to protect them from osteoporosis in low gravity, while people who go to live at the bottom of the ocean will want a different set of modifications. And people who want to be super bankers are probably going to want a different set than the people who want to be super athletes. There isn’t a best kind of human, just like there isn’t a best kind of car.

    If you’re going to worry – which I do all the time – I would worry about adult augmentation, because it will spread fast. If I were to augment a child, an embryo, it will take 20 years before they have any significant impact on society.

    But if I come up with an augmentation that improves adult intelligence, news of that will go through the internet at light speed and a lot of people will try it. And then if it’s successful in the first million people who try it, then there could be a billion people who try it.

    In 2012 you published a book entitled Regenesis, and you have talked about your lab as a centre for new technology aiming to rebuild creation to suit humans. Set that alongside your appearance and your name, and is it any wonder people accuse you of playing God?

    It’s certainly not my intention. But we’re engineers – making our world suit us is what we do. It’s what humans do. The term “playing God” is mainly used to imply that you are doing something beyond your means.

    I agree that we need to be cautious and I actually feel that humanity generally does move forward in steps. It can seem very fast and can, ultimately, happen in gigantic leaps – like landing on the moon and eliminating smallpox – but these things are done cautiously.

    Yet technology is advancing at a blistering pace.

    Einstein said our technology exceeds our humanity. I don’t buy that. I think that even when technology is going very fast, we have tried-and-tested traditional ways of reining it in. We don’t need special bans or a moratorium – we have the Environmental Protection Agency, we have the FDA. We need to think big, but also think carefully.

    See the full article here .

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  • richardmitnick 2:12 pm on September 7, 2015 Permalink | Reply
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    From phys.org: “Scientists create world’s largest protein map to reveal which proteins work together in a cell” 


    September 7, 2015
    No Writer Credit

    Scientists have uncovered tens of thousands of new protein interactions, accounting for about a quarter of all estimated protein contacts in a cell. Credit: Jovana Drinkjakovic

    A multinational team of scientists have sifted through cells of vastly different organisms, from amoebae to worms to mice to humans, to reveal how proteins fit together to build different cells and bodies.

    This tour de force of protein science, a result of a collaboration between seven research groups from three countries, led by Professor Andrew Emili from the University of Toronto’s Donnelly Centre and Professor Edward Marcotte from the University of Texas at Austin, uncovered tens of thousands of new protein interactions, accounting for about a quarter of all estimated protein contacts in a cell.

    When even a single one of these interactions is lost it can lead to disease, and the map is already helping scientists spot individual proteins that could be at the root of complex human disorders. The data will be available to researchers across the world through open access databases.

    The study comes out in Nature on September 7.

    While the sequencing of the human genome more than a decade ago was undoubtedly one of the greatest discoveries in biology, it was only the beginning of our in-depth understanding of how cells work. Genes are just blueprints and it is the genes’ products, the proteins, that do much of the work in a cell.

    Proteins work in teams by sticking to each other to carry out their jobs. Many proteins come together to form so called molecular machines that play key roles, such a building new proteins or recycling those no longer needed by literally grinding them into reusable parts. But for the vast majority of proteins, and there are tens of thousands of them in human cells, we still don’t know what they do.

    This is where Emili and Marcotte’s map comes in. Using a state-of-the-art method developed by the groups, the researchers were able to fish thousands of protein machineries out of cells and count individual proteins they are made of. They then built a network that, similar to social networks, offers clues into protein function based on which other proteins they hang out with. For example, a new and unstudied protein, whose role we don’t yet know, is likely to be involved in fixing damage in a cell if it sticks to cell’s known “handymen” proteins.

    Today’s landmark study gathered information on protein machineries from nine species that represent the tree of life: baker’s yeast, amoeba, sea anemones, flies, worms, sea urchins, frogs, mice and humans. The new map expands the number of known protein associations over 10 fold, and gives insights into how they evolved over time.

    “For me the highlight of the study is its sheer scale. We have tripled the number of protein interactions for every species. So across all the animals, we can now predict, with high confidence, more than 1 million protein interactions – a fundamentally ‘big step’ moving the goal posts forward in terms of protein interactions networks,” says Emili, who is also Ontario Research Chair in Biomarkers in Disease Management and a professor in the Department of Molecular Genetics.

    The researchers discovered that tens of thousands of protein associations remained unchanged since the first ancestral cell appeared, one billion years ago (!), preceding all of animal life on Earth.

    “Protein assemblies in humans were often identical to those in other species. This not only reinforces what we already know about our common evolutionary ancestry, it also has practical implications, providing the ability to study the genetic basis for a wide variety of diseases and how they present in different species,” says Marcotte.

    The map is already proving useful in pinpointing possible causes of human disease. One example is a newly discovered molecular machine, dubbed Commander, which consists of about a dozen individual proteins. Genes that encode some of Commander’s components had previously been found to be mutated in people with intellectual disabilities but it was not clear how these proteins worked.

    Because Commander is present in all animal cells, graduate student Fan Tu went on to disrupt its components in tadpoles, revealing abnormalities in the way brain cells are positioned during embryo development and providing a possible origin for a complex human condition.

    “With tens of thousands of other new protein interactions, our map promises to open many more lines of research into links between proteins and disease, which we are keen to explore in depth over the coming years,” concludes Dr. Emili.

    See the full article here .

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    About Phys.org in 100 Words

    Phys.org™ (formerly Physorg.com) is a leading web-based science, research and technology news service which covers a full range of topics. These include physics, earth science, medicine, nanotechnology, electronics, space, biology, chemistry, computer sciences, engineering, mathematics and other sciences and technologies. Launched in 2004, Phys.org’s readership has grown steadily to include 1.75 million scientists, researchers, and engineers every month. Phys.org publishes approximately 100 quality articles every day, offering some of the most comprehensive coverage of sci-tech developments world-wide. Quancast 2009 includes Phys.org in its list of the Global Top 2,000 Websites. Phys.org community members enjoy access to many personalized features such as social networking, a personal home page set-up, RSS/XML feeds, article comments and ranking, the ability to save favorite articles, a daily newsletter, and other options.

  • richardmitnick 7:57 am on August 25, 2015 Permalink | Reply
    Tags: , , Genetics,   

    From phys.org: “Genetic overlapping in multiple autoimmune diseases may suggest common therapies” 


    August 24, 2015
    No Writer Credit

    DNA double helix. Credit: public domain

    Scientists who analyzed the genes involved in 10 autoimmune diseases that begin in childhood have discovered 22 genome-wide signals shared by two or more diseases. These shared gene sites may reveal potential new targets for treating many of these diseases, in some cases with existing drugs already available for non-autoimmune disorders.

    Autoimmune diseases, such as type 1 diabetes, Crohn’s disease, and juvenile idiopathic arthritis, collectively affect 7 to 10 percent of the population in the Western Hemisphere.

    “Our approach did more than finding genetic associations among a group of diseases,” said study leader, Hakon Hakonarson, M.D., Ph.D., director of the Center for Applied Genomics at The Children’s Hospital of Philadelphia (CHOP). “We identified genes with a biological relevance to these diseases, acting along gene networks and pathways that may offer very useful targets for therapy.”

    The paper appears online today in Nature Medicine.

    The international study team performed a meta-analysis, including a case-control study of 6,035 subjects with automimmune disease and 10,700 controls, all of European ancestry. The study’s lead analyst, Yun (Rose) Li, an M.D./Ph.D. graduate student at the University of Pennsylvania and the Center for Applied Genomics, mentored by Hakonarson and his research team, applied highly innovative and integrative approaches in supporting the study of pathogenic roles of the genes uncovered across multiple diseases.

    The research encompassed 10 clinically distinct autoimmune diseases with onset during childhood: type 1 diabetes, celiac disease, juvenile idiopathic arthritis, common variable immunodeficiency disease, systemic lupus erythematosus, Crohn’s disease, ulcerative colitis, psoriasis, autoimmune thyroiditis and ankylosing spondylitis.

    Because many of these diseases run in families and because individual patients often have more than one autoimmune condition, clinicians have long suspected these conditions have shared genetic predispositions. Previous genome-wide association studies have identified hundreds of susceptibility genes among autoimmune diseases, largely affecting adults.

    The current research was a systematic analysis of multiple pediatric-onset diseases simultaneously. The study team found 27 genome-wide loci, including five novel loci, among the diseases examined. Of those 27 signals, 22 were shared by at least two of the autoimmune diseases, and 19 of them were shared by at least three of them.

    Many of the gene signals the investigators discovered were on biological pathways functionally linked to cell activation, cell proliferation and signaling systems important in immune processes. One of the five novel signals, near the CD40LG gene, was especially compelling, said Hakonarson, who added, “That gene encodes the ligand for the CD40 receptor, which is associated with Crohn’s disease, ulcerative colitis and celiac disease. This ligand may represent another promising drug target in treating these diseases.”

    Many of the 27 gene signals the investigators uncovered have a biological relevance to autoimmune disease processes, Hakonarson said. “Rather than looking at overall gene expression in all cells, we focused on how these genes upregulated gene expression in specific cell types and tissues, and found patterns that were directly relevant to specific diseases. For instance, among several of the diseases, we saw genes with stronger expression in B cells. Looking at diseases such as lupus or juvenile idiopathic arthritis, which feature dysfunctions in B cells, we can start to design therapies to dial down over-expression in those cells.”

    He added that “the level of granularity the study team uncovered offers opportunities for researchers to better target gene networks and pathways in specific autoimmune diseases, and perhaps to fine tune and expedite drug development by repurposing existing drugs, based on our findings.”

    More information: Meta-analysis of shared genetic architecture across ten pediatric autoimmune diseases, Nature Medicine, published online Aug. 24, 2015. doi.org/10.1038/nm.3933

    See the full article here.

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    About Phys.org in 100 Words

    Phys.org™ (formerly Physorg.com) is a leading web-based science, research and technology news service which covers a full range of topics. These include physics, earth science, medicine, nanotechnology, electronics, space, biology, chemistry, computer sciences, engineering, mathematics and other sciences and technologies. Launched in 2004, Phys.org’s readership has grown steadily to include 1.75 million scientists, researchers, and engineers every month. Phys.org publishes approximately 100 quality articles every day, offering some of the most comprehensive coverage of sci-tech developments world-wide. Quancast 2009 includes Phys.org in its list of the Global Top 2,000 Websites. Phys.org community members enjoy access to many personalized features such as social networking, a personal home page set-up, RSS/XML feeds, article comments and ranking, the ability to save favorite articles, a daily newsletter, and other options.

  • richardmitnick 4:24 pm on July 19, 2015 Permalink | Reply
    Tags: , , , Genetics,   

    From WIRED: “Chemists Invent New Letters for Nature’s Genetic Alphabet” 

    Wired logo


    Emily Singer

    Olena Shmahalo/Quanta Magazine

    DNA stores our genetic code in an elegant double helix.

    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.

    But some argue that this elegance is overrated. “DNA as a molecule has many things wrong with it,” said Steven Benner, an organic chemist at the Foundation for Applied Molecular Evolution in Florida.

    Nearly 30 years ago, Benner sketched out better versions of both DNA and its chemical cousin RNA, adding new letters and other additions that would expand their repertoire of chemical feats.

    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.

    He wondered why these improvements haven’t occurred in living creatures. Nature has written the entire language of life using just four chemical letters: G, C, A and T. Did our genetic code settle on these four nucleotides for a reason? Or was this system one of many possibilities, selected by simple chance? Perhaps expanding the code could make it better.

    Benner’s early attempts at synthesizing new chemical letters failed. But with each false start, his team learned more about what makes a good nucleotide and gained a better understanding of the precise molecular details that make DNA and RNA work. The researchers’ efforts progressed slowly, as they had to design new tools to manipulate the extended alphabet they were building. “We have had to re-create, for our artificially designed DNA, all of the molecular biology that evolution took 4 billion years to create for natural DNA,” Benner said.

    Now, after decades of work, Benner’s team has synthesized artificially enhanced DNA that functions much like ordinary DNA, if not better. In two papers published in the Journal of the American Chemical Society last month, the researchers have shown that two synthetic nucleotides called P and Z fit seamlessly into DNA’s helical structure, maintaining the natural shape of DNA. Moreover, DNA sequences incorporating these letters can evolve just like traditional DNA, a first for an expanded genetic alphabet.

    The new nucleotides even outperform their natural counterparts. When challenged to evolve a segment that selectively binds to cancer cells, DNA sequences using P and Z did better than those without.

    “When you compare the four-nucleotide and six-nucleotide alphabet, the six-nucleotide version seems to have won out,” said Andrew Ellington, a biochemist at the University of Texas, Austin, who was not involved in the study.

    Benner has lofty goals for his synthetic molecules. He wants to create an alternative genetic system in which proteins—intricately folded molecules that perform essential biological functions—are unnecessary. Perhaps, Benner proposes, instead of our standard three-component system of DNA, RNA and proteins, life on other planets evolved with just two.

    Better Blueprints for Life

    The primary job of DNA is to store information. Its sequence of letters contains the blueprints for building proteins. Our current four-letter alphabet encodes 20 amino acids, which are strung together to create millions of different proteins. But a six-letter alphabet could encode as many as 216 possible amino acids and many, many more possible proteins.

    Expanding the genetic alphabet dramatically expands the number of possible amino acids and proteins that cells can build, at least in theory. The existing four-letter alphabet produces 20 amino acids (small circle) while a six-letter alphabet could produce 216 possible amino acids. Olena Shmahalo/Quanta Magazine

    Why nature stuck with four letters is one of biology’s fundamental questions. Computers, after all, use a binary system with just two “letters”—0s and 1s. Yet two letters probably aren’t enough to create the array of biological molecules that make up life. “If you have a two-letter code, you limit the number of combinations you get,” said Ramanarayanan Krishnamurthy, a chemist at the Scripps Research Institute in La Jolla, Calif.

    On the other hand, additional letters could make the system more error prone. DNA bases come in pairs—G pairs with C and A pairs with T. It’s this pairing that endows DNA with the ability to pass along genetic information. With a larger alphabet, each letter has a greater chance of pairing with the wrong partner, and new copies of DNA might harbor more mistakes. “If you go past four, it becomes too unwieldy,” Krishnamurthy said.

    But perhaps the advantages of a larger alphabet can outweigh the potential drawbacks. Six-letter DNA could densely pack in genetic information. And perhaps six-letter RNA could take over some of the jobs now handled by proteins, which perform most of the work in the cell.

    Proteins have a much more flexible structure than DNA and RNA and are capable of folding into an array of complex shapes. A properly folded protein can act as a molecular lock, opening a chamber only for the right key. Or it can act as a catalyst, capturing and bringing together different molecules for chemical reactions.

    Adding new letters to RNA could give it some of these abilities. “Six letters can potentially fold into more, different structures than four letters,” Ellington said.

    Back when Benner was sketching out ideas for alternative DNA and RNA, it was this potential that he had in mind. According to the most widely held theory of life’s origins, RNA once performed both the information-storage job of DNA and the catalytic job of proteins. Benner realized that there are many ways to make RNA a better catalyst.

    “With just these little insights, I was able to write down the structures that are in my notebook as alternatives that would make DNA and RNA better,” Benner said. “So the question is: Why did life not make these alternatives? One way to find out was to make them ourselves, in the laboratory, and see how they work.”

    Steven Benner’s lab notebook from 1985 outlining plans to synthesize “better” DNA and RNA by adding new chemical letters. Courtesy of Steven Benner

    It’s one thing to design new codes on paper, and quite another to make them work in real biological systems. Other researchers have created their own additions to the genetic code, in one case even incorporating new letters into living bacteria. But these other bases fit together a bit differently from natural ones, stacking on top of each other rather than linking side by side. This can distort the shape of DNA, particularly when a number of these bases cluster together. Benner’s P-Z pair, however, is designed to mimic natural bases.

    One of the new papers by Benner’s team shows that Z and P are yoked together by the same chemical bond that ties A to T and C to G. (This bond is known as Watson-Crick pairing, after the scientists who discovered DNA’s structure.) Millie Georgiadis, a chemist at Indiana University-Purdue University Indianapolis, along with Benner and other collaborators, showed that DNA strands that incorporate Z and P retain their proper helical shape if the new letters are strung together or interspersed with natural letters.

    “This is very impressive work,” said Jack Szostak, a chemist at Harvard University who studies the origin of life, and who was not involved in the study. “Finding a novel base pair that does not grossly disrupt the double-helical structure of DNA has been quite difficult.”

    The team’s second paper demonstrates how well the expanded alphabet works. Researchers started with a random library of DNA strands constructed from the expanded alphabet and then selected the strands that were able to bind to liver cancer cells but not to other cells. Of the 12 successful binders, the best had Zs and Ps in their sequences, while the weakest did not.

    “More functionality in the nucleobases has led to greater functionality in nucleic acids themselves,” Ellington said. In other words, the new additions appear to improve the alphabet, at least under these conditions.

    But additional experiments are needed to determine how broadly that’s true. “I think it will take more work, and more direct comparisons, to be sure that a six-letter version generally results in ‘better’ aptamers [short DNA strands] than four-letter DNA,” Szostak said. For example, it’s unclear whether the six-letter alphabet triumphed because it provided more sequence options or because one of the new letters is simply better at binding, Szostak said.

    Benner wants to expand his genetic alphabet even further, which could enhance its functional repertoire. He’s working on creating a 10- or 12-letter system and plans to move the new alphabet into living cells. Benner’s and others’ synthetic molecules have already proved useful in medical and biotech applications, such as diagnostic tests for HIV and other diseases. Indeed, Benner’s work helped to found the burgeoning field of synthetic biology, which seeks to build new life, in addition to forming useful tools from molecular parts.

    Why Life’s Code Is Limited

    Benner’s work and that of other researchers suggests that a larger alphabet has the capacity to enhance DNA’s function. So why didn’t nature expand its alphabet in the 4 billion years it has had to work on it? It could be because a larger repertoire has potential disadvantages. Some of the structures made possible by a larger alphabet might be of poor quality, with a greater risk of misfolding, Ellington said.

    Nature was also effectively locked into the system at hand when life began. “Once [nature] has made a decision about which molecular structures to place at the core of its molecular biology, it has relatively little opportunity to change those decisions,” Benner said. “By constructing unnatural systems, we are learning not only about the constraints at the time that life first emerged, but also about constraints that prevent life from searching broadly within the imagination of chemistry.”

    The genetic code—made up of the four letters, A, T, G and C—stores the blueprint for proteins. DNA is first transcribed into RNA and then translated into proteins, which fold into specific shapes. Olena Shmahalo/Quanta Magazine

    Benner aims to make a thorough search of that chemical space, using his discoveries to make new and improved versions of both DNA and RNA. He wants to make DNA better at storing information and RNA better at catalyzing reactions. He hasn’t shown directly that the P-Z base pairs do that. But both bases have the potential to help RNA fold into more complex structures, which in turn could make proteins better catalysts. P has a place to add a “functional group,” a molecular structure that helps folding and is typically found in proteins. And Z has a nitro group, which could aid in molecular binding.

    In modern cells, RNA acts as an intermediary between DNA and proteins. But Benner ultimately hopes to show that the three-biopolymer system—DNA, RNA and proteins—that exists throughout life on Earth isn’t essential. With better-engineered DNA and RNA, he says, perhaps proteins are unnecessary.

    Indeed, the three-biopolymer system may have drawbacks, since information flows only one way, from DNA to RNA to proteins. If a DNA mutation produces a more efficient protein, that mutation will spread slowly, as organisms without it eventually die off.

    What if the more efficient protein could spread some other way, by directly creating new DNA? DNA and RNA can transmit information in both directions. So a helpful RNA mutation could theoretically be transformed into beneficial DNA. Adaptations could thus lead directly to changes in the genetic code.

    Benner predicts that a two-biopolymer system would evolve faster than our own three-biopolymer system. If so, this could have implications for life on distant planets. “If we find life elsewhere,” he said, “it would likely have the two-biopolymer system.”

    See the full article here.

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  • richardmitnick 1:40 pm on April 22, 2015 Permalink | Reply
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    From Princeton: “Decoding the Cell’s Genetic Filing System (Nature Chemistry)” 

    Princeton University
    Princeton University

    April 22, 2015
    Tien Nguyen

    Source: Nature Chemistry

    A fully extended strand of human DNA measures about five feet in length. Yet it occupies a space just one-tenth of a cell by wrapping itself around histones—spool-like proteins—to form a dense hub of information called chromatin.

    Access to these meticulously packed genes is regulated by post-translational modifications, chemical changes to the structure of histones that act as on-off signals for gene transcription. Mistakes or mutations in histones can cause diseases such as glioblastoma, a devastating pediatric brain cancer.

    Researchers at Princeton University have developed a facile method to introduce non-native chromatin into cells to interrogate these signaling pathways. Published on April 6 in the journal Nature Chemistry, this work is the latest chemical contribution from the Muir lab towards understanding nature’s remarkable information indexing system.

    Tom Muir, the Van Zandt Williams, Jr. Class of ’65 Professor of Chemistry, began investigating transcriptional pathways in the so-called field of epigenetics almost a decade earlier. Deciphering such a complex and dynamic system posed a formidable challenge, but his research lab was undeterred. “It’s better to fail at something important than to succeed at something trivial,” he said.

    Muir recognized the value of introducing chemical approaches to epigenetics to complement early contributions that came mainly from molecular biologists and geneticists. If epigenetics was like a play, he said, molecular biology and genetics could identify the characters but chemistry was needed to understand the subplots.

    These subplots, or post-translational modifications of histones, of which there are more than 100, can occur cooperatively and simultaneously. Traditional methods to probe post-translational modifications involved synthesizing modified histones one at a time, which was a very slow process that required large amounts of biological material.

    Last year, the Muir group introduced a method that would massively accelerate this process. The researchers generated a library of 54 nucleosomes—single units of chromatin, like pearls on a necklace—encoded with DNA-barcodes, unique genetic tags that can be easily identified. Published in the journal Nature Methods, the high throughput method required only microgram amounts of each nucleosome to run approximately 4,500 biochemical assays.

    “The speed and sensitivity of the assay was shocking,” Muir said. Each biochemical assay involved treatment of the DNA-barcoded nucleosome with a writer, reader or nuclear extract, to reveal a particular binding preference of the histone. The products were then isolated using a technique called chromatin immunoprecipitation and characterized by DNA sequencing, essentially an ordered readout of the nucleotides.

    “There have been incredible advances in genetic sequencing over the last 10 years that have made this work possible,” said Manuel Müller, a postdoctoral researcher in the Muir lab and co-author on the Nature Methods article.

    Schematic of approach using split inteins

    With this method, researchers could systematically interrogate the signaling system to propose mechanistic pathways. But these mechanistic insights would remain hypotheses unless they could be validated in vivo, meaning inside the cellular environment.

    The only method for modifying histones in vivo was extremely complicated and specific, said Yael David, a postdoctoral researcher in the Muir lab and lead author on the recent Nature Chemistry study that demonstrated a new and easily customizable approach.

    The method relied on using ultra-fast split inteins, protein fragments that have a great affinity for one another. First, one intein fragment was attached to a modified histone, by encoding it into a cell. Then, the other intein fragment was synthetically fused to a label, which could be a small protein tag, fluorophore or even an entire protein like ubiquitin.

    Within minutes of being introduced into the cell, the labeled intein fragment bound to the histone intein fragment. Then like efficient and courteous matchmakers, the inteins excised themselves and created a new bond between the label and modified histone. “It’s really a beautiful way to engineer proteins in a cell,” David said.

    Regions of the histone may be loosely or tightly packed, depending on signals from the cell indicating whether or not to transcribe a gene. By gradually lowering the amount of labeled intein introduced, the researchers could learn about the structure of chromatin and tease out which areas were more accessible than others.

    Future plans in the Muir lab will employ these methods to ask specific biological questions, such as whether disease outcomes can be altered by manipulating signaling pathway. “Ultimately, we’re developing methods at the service of biological questions,” Muir said.

    Read the articles:

    Nguyen, U.T.T.; Bittova, L.; Müller, M.; Fierz, B.; David, Y.; Houck-Loomis, B.; Feng, V.; Dann, G.P.; Muir, T.W. Accelerated chromatin biochemistry using DNA-barcoded nucleosome libraries. Nature Methods, 2014, 11, 834.

    David, Y.; Vila-Perelló, M; Verma, S.; Muir, T.W. Chemical tagging and customizing of cellular chromatin states using ultrafast trans-splicing inteins. Nature Chemistry, Advance online publication, April 6, 2015.

    See the full article here.

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    Princeton University Campus

    About Princeton: Overview

    Princeton University is a vibrant community of scholarship and learning that stands in the nation’s service and in the service of all nations. Chartered in 1746, Princeton is the fourth-oldest college in the United States. Princeton is an independent, coeducational, nondenominational institution that provides undergraduate and graduate instruction in the humanities, social sciences, natural sciences and engineering.

    As a world-renowned research university, Princeton seeks to achieve the highest levels of distinction in the discovery and transmission of knowledge and understanding. At the same time, Princeton is distinctive among research universities in its commitment to undergraduate teaching.

    Today, more than 1,100 faculty members instruct approximately 5,200 undergraduate students and 2,600 graduate students. The University’s generous financial aid program ensures that talented students from all economic backgrounds can afford a Princeton education.

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  • richardmitnick 5:33 pm on April 20, 2015 Permalink | Reply
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    From UCSD: “Genetics Overlap Found Between Alzheimer’s Disease and Cardiovascular Risk Factors” 

    UC San Diego bloc

    UC San Diego

    April 16, 2015
    Scott LaFee

    An international team of scientists, led by researchers at University of California, San Diego School of Medicine, have found genetic overlap between Alzheimer’s disease (AD) and two significant cardiovascular disease risk factors: high levels of inflammatory C-reactive protein (CRP) and plasma lipids or fats. The findings, based upon genome-wide association studies involving hundreds of thousands of individuals, suggest the two cardiovascular phenotypes play a role in AD risk – and perhaps offer a new avenue for potentially delaying disease progression.

    The findings are published in current online issue of Circulation.

    “For many years we have known that high levels of cholesterol and high levels of inflammation are associated with increased risks for Alzheimer’s disease,” said study co-author Paul M. Ridker, MD, MPH, the Eugene Braunwald Professor of Medicine at Harvard Medical School and director of the Center for Cardiovascular Disease Prevention at Brigham and Women’s Hospital. “The current work finds that specific genetic signals explain a part of these relationships. We now need to characterize the function of these genetic signals and see whether they can help us to design better trials evaluating inflammation inhibition as a possible method for Alzheimer’s treatment.”

    The researchers used summary statistics from genome-wide association studies of more than 200,000 individuals, looking for overlap in single nucleotide polymorphisms (SNPs) associated with clinically diagnosed AD and CRP and the three components of total cholesterol: high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TG). SNPs are fragments of DNA sequence that commonly vary among individuals within a population.

    They found up to a 50-fold enrichment of AD SNPs for different levels of association with CRP, LDL, HDL and TG, which then lead to identification of 55 loci – specific locations on a gene, DNA sequence or chromosome – linked to increased AD risk. The researchers next conducted a meta-analysis of these 55 variants across four independent AD study cohorts, encompassing almost 145,000 persons with AD and healthy controls, revealing two genome-wide significant variants on chromosomes 4 and 10. The two identified genes – HS3ST1 and ECHDC3 – were not previously associated with AD risk.

    “Our findings indicate that a subset of genes involved with elevated plasma lipid levels and inflammation may also increase the risk for developing AD. Elevated levels of plasma lipids and inflammation can be modified with treatment, which means it could be possible to identify and therapeutically target individuals at increased risk for developing cardiovascular disease who are also at risk for developing Alzheimer’s disease,” said Rahul S. Desikan, MD, PhD, research fellow and radiology resident at the UC San Diego School of Medicine and the study’s first author.

    If so, the research may have significant ramifications. Late-onset AD is the most common form of dementia, affecting an estimated 30 million persons worldwide – a number that is expected to quadruple over the next 40 years. The societal costs, from medical to lost productivity, are staggering. The 2010 World Alzheimer Report estimated total annual costs at $606 billion.

    “Currently, there are no disease modifying therapies and much attention has been focused upon prevention and early diagnosis,” said Ole A. Andreassen, MD, PhD, a senior co-author and professor of biological psychiatry at the University of Oslo in Norway. “Delaying dementia onset by even just two years could potentially lower the worldwide prevalence of AD by more than 22 million cases over the next four decades, resulting in significant societal savings.”

    Senior author Anders M. Dale, PhD, professor of neurosciences and radiology and director of the Center for Translational Imaging and Precision Medicine at UC San Diego, said further research will be needed: “Careful and considerable effort will be required to further characterize the novel candidate genes detected in this study and to detect the functional variants responsible for the association of these loci with Alzheimer’s risk. It will also be important to understand whether these genes, in combination with other known markers such as brain imaging, cerebrospinal fluid measurements and APOE E4 status, can improve the prediction of disease risk in AD.”

    Co-authors include Linda K. McEvoy, and David S. Karow, UCSD Department of Radiology; Andrew J. Schork, UCSD Department of Cognitive Science; Yunpeng Wang, UCSD Department of Neurosciences and NORMENT and University of Oslo; Wesley K. Thompson, UCSD Department of Psychiatry; Abbas Dehghan, M. Arfan Ikram, and Sven J. van der Lee, Erasmus Medical Center, Netherlands; Daniel I. Chasman, Brigham and Women’s Hospital; Dominic Holland, UCSD Department of Neurosciences; Chi-Hua Chen, UCSD Department of Radiology and NORMENT; James B. Brewer, UCSD departments of Radiology and Neurosciences; Christopher P. Hess, UCSF; Julie Williams, Rebecca Sims, and Michael C. O’Donovan, Cardiff University School of Medicine, UK; Seung Hoan Choi, Boston University; Joshua C. Bis, and Cornelia M. van Duijn, University of Washington; Vilmundur Gudnason, and Anita L.DeStefano, University of Iceland; Bruce M. Psaty, NHLBI; Lenore Launer, NIA; Sudha Seshadri, NHLBI and Boston University School of Medicine; Margaret A. Pericak-Vance, University of Miami; Richard Mayeux, Columbia University; Jonathan L. Haines, Case Western University; Lindsay A. Farrer, Boston University Schools of Medicine and Public Health; John Hardy, University College London; Ingun Dina Ulstein and Dag Aarsland, Oslo University Hospital; Tormod Fladby, University of Oslo; Linda R. White, and Sigrid B. Sando, Norwegian University of Science and Technology and Trondheim University Hospital; Arvid Rongve, Haugesund Hospital; Aree Witoelar, NORMENT; Srdjan Djurovic, University of Bergen, Norway; Bradley T. Hyman, Massachusetts General Hospital; Jon Snaedal, University Hospital Reykjavik; Stacy Steinberg, and Hreinn Stefansson, deCODE Genetics; Kari Stefansson, deCODE Genetics and University of Iceland; and Gerard D. Schellenberg, University of Pennsylvania.

    Funding for this research came, in part, from the National Institutes of Health (K02 NS067427; T32 EB005970; R01GM104400-01A; R01MH100351; AG033193 and U0149505), the Research Council of Norway, the South East Norway Health Authority, Norwegian Health Association and the KG Jebsen Foundation.

    See the full article here.

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    UC San Diego Campus

    The University of California, San Diego (also referred to as UC San Diego or UCSD), is a public research university located in the La Jolla area of San Diego, California, in the United States.[12] The university occupies 2,141 acres (866 ha) near the coast of the Pacific Ocean with the main campus resting on approximately 1,152 acres (466 ha).[13] Established in 1960 near the pre-existing Scripps Institution of Oceanography, UC San Diego is the seventh oldest of the 10 University of California campuses and offers over 200 undergraduate and graduate degree programs, enrolling about 22,700 undergraduate and 6,300 graduate students. UC San Diego is one of America’s Public Ivy universities, which recognizes top public research universities in the United States. UC San Diego was ranked 8th among public universities and 37th among all universities in the United States, and rated the 18th Top World University by U.S. News & World Report ‘s 2015 rankings.

  • richardmitnick 4:04 pm on April 16, 2015 Permalink | Reply
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    From Quanta: “How Structure Arose in the Primordial Soup” 

    Quanta Magazine
    Quanta Magazine

    Life’s first epoch saw incredible advances — cells, metabolism and DNA, to name a few. Researchers are resurrecting ancient proteins to illuminate the biological dark ages.

    April 16, 2015
    Emily Singer

    Olena Shmahalo/Quanta Magazine

    About 4 billion years ago, molecules began to make copies of themselves, an event that marked the beginning of life on Earth. A few hundred million years later, primitive organisms began to split into the different branches that make up the tree of life. In between those two seminal events, some of the greatest innovations in existence emerged: the cell, the genetic code and an energy system to fuel it all. All three of these are essential to life as we know it, yet scientists know disappointingly little about how any of these remarkable biological innovations came about.

    “It’s very hard to infer even the relative ordering of evolutionary events before the last common ancestor,” said Greg Fournier, a geobiologist at the Massachusetts Institute of Technology. Cells may have appeared before energy metabolism, or perhaps it was the other way around. Without fossils or DNA preserved from organisms living during this period, scientists have had little data to work from.

    Fournier is leading an attempt to reconstruct the history of life in those evolutionary dark ages — the hundreds of millions of years between the time when life first emerged and when it split into what would become the endless tangle of existence.

    He is using genomic data from living organisms to infer the DNA sequence of ancient genes as part of a growing field known as paleogenomics. In research published online in March in the Journal of Molecular Evolution, Fournier showed that the last chemical letter added to the code was a molecule called tryptophan — an amino acid most famous for its presence in turkey dinners. The work supports the idea that the genetic code evolved gradually.

    Using similar methods, he hopes to decipher the temporal order of more of the code — determining when each letter was added to the genetic alphabet — and to date key events in the origins of life, such as the emergence of cells.

    Dark Origins

    Life emerged so long ago that even the rock formations covering the planet at that time have been destroyed — and with them, most chemical and geological clues to early evolution. “There’s a huge chasm between the origins of life and the last common ancestor,” said Eric Gaucher, a biologist at the Georgia Institute of Technology in Atlanta.

    The stretch of time between the origins of life and the last universal common ancestor saw a series of remarkable innovations — the origins of cells, metabolism and the genetic code. But scientists know little about when they happened or the order in which they occurred. Olena Shmahalo/Quanta Magazine

    Scientists do know that at some point in that time span, living creatures began using a genetic code, a blueprint for making complex proteins. It is those proteins that carry out the vital functions of the cell. (The structure of DNA and RNA also enables genetic information to be replicated and passed on from generation to generation, but that’s a separate process from the creation of proteins.) The components of the code and the molecular machinery that assembles them “are some of the oldest and most universal aspects of cells, and biologists are very interested in understanding the mechanisms by which they evolved,” said Paul Higgs, a biophysicist at McMaster University in Hamilton, Ontario.

    How the code came into being presents a chicken-and-egg problem. The key players in the code — DNA, RNA, amino acids, and proteins — are chemically complicated structures that work together to make proteins. But in modern cells, proteins are used to make the components of the code. So how did a highly structured code emerge?

    Most researchers believe that the code began simply with basic proteins made from a limited alphabet of amino acids. It then grew in complexity over time, as these proteins learned to make more sophisticated molecules. Eventually, it developed into a code capable of creating all the diversity we see today. “It’s long been hypothesized that life’s ‘standard alphabet’ of 20 amino acids evolved from a simpler, earlier alphabet, much as the English alphabet has accumulated extra letters over its history,” said Stephen Freeland, a biologist at the University of Maryland, Baltimore County.

    The earliest amino acid letters in the code were likely the simplest in structure, those that can be made from purely chemical means, without the assistance of a protein helper. (For example, the amino acids glycine, alanine and glutamic acid have been found on meteorites, suggesting they can form spontaneously in a variety of environments.) These are like the letters A, E and S — primordial units that served as the foundation for what came later.

    Tryptophan, in comparison, has a complex structure and is comparatively rare in the protein code, like a Y or Z, leading scientists to theorize that it was one of the latest additions to the code.

    That chemical evidence is compelling, but circumstantial. Enter Fournier. He suspected that by extending his work on paleogenomics, he would be able to prove tryptophan’s status as the last letter added to the code.

    The Last Letter

    Scientists have been reconstructing ancient proteins for more than a decade, primarily to figure out how ancient proteins differed from modern ones — what they looked like and how they functioned. But these efforts have focused on the period of evolution after the last universal common ancestor (or LUCA, as researchers call it). Fournier’s work delves further back than any other previous efforts. To do so, he had to move beyond the standard application of comparative genomics, which analyzes the differences between branches on the tree of life. “By definition, anything pre-LUCA lies beyond the deepest split in the tree,” he said.

    Fournier started with two related proteins, TrpRS (tryptophanyl tRNA synthetase) and TyrRS (tyrosyl tRNA synthetase), which help decode RNA letters into the amino acids tryptophan and tyrosine. TrpRS and TyrRS are more closely related to each other than to any other protein, indicating that they evolved from the same ancestor protein. Sometime before LUCA, that parent protein mutated slightly to produce these two new proteins with distinct functions. Fournier used computational techniques to decipher what that ancestral protein must look like.

    Greg Fournier, a geobiologist at MIT, is searching for the origins of the genetic code. Helen Hill

    He found that the ancestral protein has all the amino acids but tryptophan, suggesting that its addition was the finishing touch to the genetic code. “It shows convincingly that tryptophan was the last amino acid added, as has been speculated before but not really nailed as has been done here,” said Nigel Goldenfeld, a physicist at the University of Illinois, Urbana-Champaign, who was not involved in the study.

    Fournier now plans to use tryptophan as a marker to date other major pre-LUCA events such as the evolution of metabolism, cells and cell division, and the mechanisms of inheritance. These three processes form a sort of biological triumvirate that laid the foundation for life as we know it today. But we know little about how they came into existence. “If we understand the order of those basic steps, it creates an arrow pointing to possible scenarios for the origins of life,” Fournier said.

    For example, if the ancestral proteins involved in metabolism lack tryptophan, some form of metabolism probably evolved early. If proteins that direct cell division are studded with tryptophan, it suggests those proteins evolved comparatively late.

    Different models for the origins of life make different predictions for which of these three processes came first. Fournier hopes his approach will provide a way to rule out some of these models. However, he cautions that it won’t definitively sort out the timing of these events.

    Fournier plans to use the same techniques to figure out the order in which other amino acids were added to the code. “It really reinforces the idea that evolution of the code itself was a progressive process,” said Paul Schimmel, a professor of molecular and cell biology at the Scripps Research Institute, who was not involved in the study. “It speaks to the refinement and subtlety that nature was using to perfect these proteins and the diversity it needed to form this vast tree of life.”

    See the full article here.

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    Formerly known as Simons Science News, Quanta Magazine is an editorially independent online publication launched by the Simons Foundation to enhance public understanding of science. Why Quanta? Albert Einstein called photons “quanta of light.” Our goal is to “illuminate science.” At Quanta Magazine, scientific accuracy is every bit as important as telling a good story. All of our articles are meticulously researched, reported, edited, copy-edited and fact-checked.

  • 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.

    Please help promote STEM in your local schools.

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    Stem Education Coalition

    NOVA is the highest rated science series on television and the most watched documentary series on public television. It is also one of television’s most acclaimed series, having won every major television award, most of them many times over.

  • richardmitnick 10:50 am on March 19, 2015 Permalink | Reply
    Tags: , Genetics, ,   

    From Rockefeller: “Scientists pinpoint molecule that controls stem cell plasticity by boosting gene expression” 

    Rockefeller U bloc

    Rockefeller University

    March 19, 2015
    Zach Veilleux | 212-327-8982

    Glowing and growing: Researchers made stem cells fluoresce green (at the base of hair follicles above) by labeling their super-enhancers, regions of the genome bound by gene-amplifying proteins. It appears one such protein, Sox9, leads the activation of super-enhancers that boost genes associated with stem cell plasticity.No mage credit

    Stem cells can have a strong sense of identity. Taken out of their home in the hair follicle, for example, and grown in culture, these cells remain true to themselves. After waiting in limbo, these cultured cells become capable of regenerating follicles and other skin structures once transplanted back into skin. It’s not clear just how these stem cells — and others elsewhere in the body — retain their ability to produce new tissue and heal wounds, even under extraordinary conditions.

    New research at Rockefeller University has identified a protein, Sox9, that takes the lead in controlling stem cell plasticity. In a paper published Wednesday (March 18) in Nature, the team describes Sox9 as a “pioneer factor” that breaks ground for the activation of genes associated with stem cell identity in the hair follicle.

    “We found that in the hair follicle, Sox9 lays the foundation for stem cell plasticity. First, Sox9 makes the genes needed by stem cells accessible, so they can become active. Then, Sox9 recruits other proteins that work together to give these “stemness” genes a boost, amplifying their expression,” says study author Elaine Fuchs, Rebecca C. Lancefield Professor, Robin Chemers Neustein Laboratory of Mammalian Cell Biology and Development. “Without Sox9, this process never happens, and hair follicle stem cells cannot survive.”

    Sox9 is a type of protein called a transcription factor, which can act like a volume dial for genes. When a transcription factor binds to a segment of DNA known as an enhancer, it cranks up the activity of the associated gene. Recently, scientists identified a less common, but more powerful version: the super-enhancer. Super-enhancers are much longer pieces of DNA, and host large numbers of cell type-specific transcription factors that bind cooperatively. Super-enhancers also contain histones, DNA-packaging proteins, that harbor specific chemical groups — epigenetic marks — that make genes they are associated with accessible so they can be expressed.

    Using an epigenetic mark associated specifically with the histones of enhancers, first author Rene Adam, a graduate student in the lab, and colleagues, identified 377 of these high-powered gene-amplifying regions in hair follicle stem cells. The majority of these super-enhancers were bound by at least five transcription factors, often including Sox9. Then, they compared the stem cell super-enhancers to those of short-lived stem cell progeny, which have begun to choose a fate, and so lost the plasticity of stem cells. These two types of cells shared only 32 percent of their superenhancers, suggesting these regions played an important role in skin cell identity. By switching off super-enhancers associated with stem cell genes, these genes were silenced while new super-enhancers were being activated to turn on hair genes.

    To better understand these dynamics, the researchers took a piece of a super-enhancer, which they called an “epicenter,” where all the stem cell transcription factors bind, and they linked it to a gene that glowed green whenever the transcription factors were present. In living mice, all the hair follicle stem cells glowed green, but surprisingly, the green gene turned off when the stem cells were taken from the follicle and placed in culture. When they put the cells back into living skin, the green glow returned.

    Another clue came from experiments performed by Hanseul Yang, another student in the lab. By examining the new super-enhancers that were gained when the stem cells were cultured, they learned that these new super-enhancers bound transcription factors that were known to be activated during wound-repair. When they used one of these epicenters to drive the green gene, the green glow appeared in culture, but not in skin. When they wounded the skin, then the green glow switched on.

    “We were learning that some super-enhancers are specifically activated in the stem cells within their native niche, while other super-enhancers specifically switch on during injury,” explained Adam. “By shifting epicenters, you can shift from one cohort of transcription factors to another to adapt to different environments. But we still needed to determine what was controlling these shifts.”

    The culprit turned out to be Sox9, the only transcription factor expressed in both living tissue and culture. Further experiments confirmed Sox9’s importance by showing, for example, that removing it spelled death for stem cells, while expressing it in the epidermis gave the skin cells features of hair follicle stem cells. These powers seemed to be special to Sox9, placing it atop the hierarchy of transcription factors in the stem cells. Sox9 is one of only a few pioneer factors known in biology which can initiate such dramatic changes in gene expression.

    “Importantly, we link this pioneer factor to super-enhancer dynamics, giving these domains a ‘one-two punch’ in governing cell identity. In the case of stem cell plasticity, Sox9 appears to be the lead factor that activates the super-enhancers that amplify genes associated with stemness,” Fuchs says. “These discoveries offer new insights into the way in which stem cells choose their fates and maintain plasticity while in transitional states, such as in culture or when repairing wounds.”

    See the full article here.

    Please help promote STEM in your local schools.

    STEM Icon

    Stem Education Coalition

    Rockefeller U Campus

    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.

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