Tagged: DNA Toggle Comment Threads | Keyboard Shortcuts

  • richardmitnick 1:38 pm on January 9, 2017 Permalink | Reply
    Tags: DNA, , Skeletal muscle mass,   

    U Aberdeen: “Gene could play role in body’s muscle mass” 

    U Aberdeen bloc

    University of Aberdeen

    09 January 2017
    Euan Wemyss
    e.wemyss@abdn.ac.uk

    1
    Scientists at the University of Aberdeen identify gene which could play role in determining muscle mass. No image credit.

    “Our research suggests this gene could play a role in regulating muscle mass and the fact that drugs have already been developed to target the gene gives us an obvious focus for further research”
    Dr Arimantas Lionikas

    Scientists have identified a gene they think could play a role in determining a person’s muscle mass – which is linked to a number of health factors, including how long someone lives.

    Previous studies have shown a link between muscle mass and life expectancy in elderly people.

    Muscle is the most abundant tissue in the body and enables many functions from allowing us to move around to allowing us to breathe.

    The amount of skeletal muscle mass each person has can vary significantly.

    Skeletal muscle mass can be increased if a person undertakes strength exercise but genetic factors play an equally important role in determining how much muscle mass a person can have.

    Now, scientists at the University of Aberdeen, led by Dr Arimantas Lionikas, have identified a gene that appears to affect muscle mass in mice. The findings have been published in Nature Genetics.

    The same gene has previously been linked with the spread of cancer and drugs have been developed to target it.

    The team hope to study these drugs further to understand their effects on muscle tissue. If there are different drugs targeting the same gene, the research could uncover which drug has the less negative effect on muscle mass.

    “Skeletal muscle mass is incredibly important in humans, especially as they get older. We have already seen in older adults that statistically, those with lower muscle mass are more likely to die at a younger age.

    “Our research suggests this gene could play a role in regulating muscle mass and the fact that drugs have already been developed to target the gene gives us an obvious focus for further research.”

    See the full article here .

    Please help promote STEM in your local schools.

    STEM Icon

    Stem Education Coalition

    U Aberdeen Campus

    Founded in 1495 by William Elphinstone, Bishop of Aberdeen and Chancellor of Scotland, the University of Aberdeen is Scotland’s third oldest and the UK’s fifth oldest university.

    William Elphinstone established King’s College to train doctors, teachers and clergy for the communities of northern Scotland, and lawyers and administrators to serve the Scottish Crown. Much of the King’s College still remains today, as do the traditions which the Bishop began.

    King’s College opened with 36 staff and students, and embraced all the known branches of learning: arts, theology, canon and civil law. In 1497 it was first in the English-speaking world to create a chair of medicine. Elphinstone’s college looked outward to Europe and beyond, taking the great European universities of Paris and Bologna as its model.
    Uniting the Rivals

    In 1593, a second, Post-Reformation University, was founded in the heart of the New Town of Aberdeen by George Keith, fourth Earl Marischal. King’s College and Marischal College were united to form the modern University of Aberdeen in 1860. At first, arts and divinity were taught at King’s and law and medicine at Marischal. A separate science faculty – also at Marischal – was established in 1892. All faculties were opened to women in 1892, and in 1894 the first 20 matriculated female students began their studies. Four women graduated in arts in 1898, and by the following year, women made up a quarter of the faculty.

    Into our Sixth Century

    Throughout the 20th century Aberdeen has consistently increased student recruitment, which now stands at 14,000. In recent years picturesque and historic Old Aberdeen, home of Bishop Elphinstone’s original foundation, has again become the main campus site.

    The University has also invested heavily in medical research, where time and again University staff have demonstrated their skills as world leaders in their field. The Institute of Medical Sciences, completed in 2002, was designed to provide state-of-the-art facilities for medical researchers and their students. This was followed in 2007 by the Health Sciences Building. The Foresterhill campus is now one of Europe’s major biomedical research centres. The Suttie Centre for Teaching and Learning in Healthcare, a £20m healthcare training facility, opened in 2009.

     
  • richardmitnick 10:17 am on January 9, 2017 Permalink | Reply
    Tags: 16S rRNA sequencing, Archaea, DNA, , , Polymerase chain reaction, Prokaryotes, The Never-Ending Quest to Rewrite the Tree of Life   

    From NOVA: “The Never-Ending Quest to Rewrite the Tree of Life” 

    PBS NOVA

    NOVA

    04 Jan 2017
    Carrie Arnold

    The bottom of the ocean is one of the most mysterious places on the planet, but microbiologist Karen Lloyd of the University of Tennessee, Knoxville, wanted to go deeper than that. In 2010, she was a postdoc at Aarhus University in Denmark, and Lloyd wanted to see what microbes were living more than 400 feet beneath the sea floor.

    Like nearly all microbiologists doing this type of census, she relied on 16S rRNA sequencing to determine who was there. Developed by microbiologist Carl Woese in the late 1970s, the technique looks for variation in the 16S rRNA gene, one that’s common to all organisms (it’s key to turning DNA into protein, one of life’s of the most fundamental processes). When Lloyd compared what she had seen under the microscope to what her sequencing data said, however, she knew her DNA results were missing a huge portion of the life hidden underneath the ocean.

    “I had two problems with just 16S sequencing. One, I knew it would miss organisms, and two, it’s not good for understanding small differences between microbes,” Lloyd says.

    1
    Scientists use heat maps like these to visualize the diversity of bacteria in various environments. Credits below.

    Technology had made gene sequencing much quicker and easier compared to when Woese first started his work back in the 1970s, but the principle remained the same. The 16S rRNA gene codes for a portion of the machinery used by prokaryotes to make protein, which is a central activity in the cell. All microbes have a copy of this gene, but different species have slightly different copies. If two species are closely related, their 16S rRNA sequences will be nearly identical; more distantly related organisms will have a greater number of differences. It not only gave researchers a way to quantify evolutionary relationships between species, Woese’s work also revealed an entirely new branch on the tree of life—the archaea, a group of microscopic organisms distinct from bacteria.

    Woese’s success in using 16S rRNA to rewrite the tree of life no doubt encouraged its widespread use. But as Lloyd and other scientists began to realize, some microbes carry a version that is significantly different from that seen in other bacteria or archaea. Since biologists depended on this similarity to identify an organism, they began to realize that they were leaving out potentially significant portions of life from their investigations.

    These concerns culminated approximately ten years ago during a period when sequencing technologies were rapidly accelerating. During this time, researchers figured out how to prepare DNA for sequencing without needing to know anything about the organism you were studying. At the same time, scientists invented a strategy to isolate single cells. At her lab at the Joint Genome Institute outside San Francisco, microbiologist Tanja Woyke put these two strategies together to sequence the genomes of individual microbial cells. Meanwhile, Jill Banfield, across the bay at the University of California, Berkeley, used a different approach called metagenomics that sequenced genes from multiple species at once, and used computer algorithms to reconstruct each organism’s genome. Over the past several years, their work has helped illuminate the massive amount of microbial dark matter that comprises life on Earth.

    “These two strategies really complement each other. They have opened up our ability to see the true diversity of microbial life,” says Roger Lasken, a microbial geneticist at the J. Craig Venter Institute.

    Microbial Dark Matter

    When Woese sequenced the 16S genes of the microbes that would come to be known as archaea, they were completely different from most of the other bacterial sequences he had accumulated. They lacked a true nucleus, like other bacteria, but their metabolisms were completely different. These microbes also tended to favor extreme environments, such as those at high temperatures (hot springs and hydrothermal vents), high salt concentrations, or high acidity. Sensing their ancient origins, Woese named these microbes the archaea, and gave them their own branch on the tree of life.

    Woese did all of his original sequencing by hand, a laborious process that took years. Later, DNA sequencing machines greatly simplified the work, although it still required amplifying the small amount of DNA present using a technique known as polymerase chain reaction, or PCR, before sequencing. The utility of 16S sequencing soon made the technique one of the mainstays of the microbiology lab, along with the Petri dish and the microscope.

    The method uses a set of what’s known as universal primers—short strands of RNA or DNA that help jump start the duplication of DNA—to make lots of copies of the 16S gene so it can be sequenced. The primers bound to a set of DNA sequences flanking the 16S gene that were thought to be common to all organisms. This acted like a set of bookends to identify the region to be copied by PCR. As DNA sequencing technology improved, researchers began amplifying and sequencing 16S genes in environmental samples as a way of identifying the microbes present without the need to grow them in the lab. Since scientists have only been able to culture about one in 100 microbial species, this method opened broad swaths of biodiversity that would otherwise have remained invisible.

    “We didn’t know that these deep branches existed. Trying to study life from just 16S rRNA sequences is like trying to understand all animals by visiting a zoo,” says Lionel Guy, a microbiologist from Uppsala University in Sweden.


    Access mp4 video here .
    Discover how to interpret and create evolutionary trees, then explore the tree of life in NOVA’s Evolution Lab.

    It didn’t take long, however, for scientists to realize the universal primers weren’t nearly as universal as researchers had hoped. The use of the primers rested on the assumption that all organisms, even unknown ones, would have similar DNA sequences surrounding the 16S rRNA gene. But that meant that any true oddballs probably wouldn’t have 16S rRNA sequences that matched the universal primers—they would remain invisible. These uncultured, unsequenced species were nicknamed “microbial dark matter” by Stanford University bioengineer and physicist Stephen Quake in a 2007 PNAS paper.

    The name, he says, is analogous to dark matter in physics, which is invisible but thought to make up the bulk of the universe. “It took DNA technology to realize the depth of the problem. I mean, holy crap, there’s a lot more out there than we can discover,” Quake says.

    Quake’s snappy portmanteau translated into the Microbial Dark Matter project—an ongoing quest in microbiology, led by Woyke, to understand the branches on the tree of life that remain shrouded in mystery by isolating DNA from single bacterial and archaeal cells. These microbial misfits intrigued Lloyd as well, and she believed the subsurface had many more of them than anyone thought. Her task was to find them.

    “We had no idea what was really there, but we knew it was something,” Lloyd says.

    To solve her Rumsfeldian dilemma of identifying both her known and unknown unknowns, Lloyd needed a DNA sequencing method that would allow her to sequence the genomes of the microbes in her sample without any preconceived notions of what they looked like. As it turns out, a scientist in New Haven, Connecticut was doing just that.

    Search for Primers

    In the 1990s, Roger Lasken had recognized the problems with traditional 16S rRNA and other forms of sequencing. Not only did you need to know something about the DNA sequence ahead of time in order to make enough genetic material to be sequenced, you also needed a fairly large sample. The result was a significant limitation in the types of material that could be sequenced. Lasken wanted to be able to sequence the genome of a single cell without needing to know anything about it.

    Then employed at the biotech firm Molecular Staging, Lasken began work on what he called multiple displacement amplification (MDA). He built on a recently discovered DNA polymerase (the enzyme that adds nucleotides, one by one, to a growing piece of DNA) called φ29 DNA polymerase. Compared to the more commonly used Taq polymerase, the φ29 polymerase created much longer strands of DNA and could operate at much cooler temperatures. Scientists had also developed random primers, small pieces of randomly generated DNA. Unlike the universal primers, which were designed to match specific DNA sequences 20–30 nucleotides in length, random primers were only six nucleotides long. This meant they were small enough to match pieces of DNA on any genome. With enough random primers to act as starting points for the MDA process, scientists could confidently amplify and sequence all the genetic material in a sample. The bonus inherent in the random primers was that scientists didn’t need to know anything about the sample they were sequencing in order to begin work.

    “For the first time, you didn’t need to culture an organism or amplify its DNA to sequence it,” he says.

    The method had only been tested on relatively small pieces of DNA. Lasken’s major breakthrough was making the system work for larger chromosomes, including those in humans, which was published in 2002 in PNAS. Lasken was halfway to his goal—his next step was figuring out how to do this in a single bacterium, which would enable researchers to sequence any microbial cell they found. In 2005, Lasken and colleagues managed to isolate a single E. coli cell and sequence its 16S rRNA gene using MDA. It was a good proof of principle that the system worked, but to understand the range and depth of microbial biodiversity, researchers like Tanja Woyke, the microbiologist at the Joint Genome Institute, needed to look at the entire genome of a single cell. In theory, the system should work neatly: grab a single cell, amplify its DNA, and then sequence it. But putting all of the steps together and working on the kinks in the system would require years of work.

    Woyke had spent her postdoc at the Joint Genome Institute sequencing DNA from samples not grown in the lab, but drawn directly from the environment, like a scoop of soil. At the time, she was using metagenomics, which amplified and sequenced DNA directly from environmental samples, yielding millions of As, Ts, Gs, and Cs from even a thimble of dirt. Woyke’s problem was determining which genes belonged to which microbe, a key step in assembling a complete genome. Nor was she able to study different strains of the same microbe that were present in a sample because their genomes were just too similar to tell apart using the available sequencing technology. What’s more, the sequences from common species often completely drowned out the data from more rare ones.

    “I kept thinking to myself, wouldn’t it be nice to get the entire genome from just a single cell,” Woyke says. Single-cell genomics would enable her to match a genome and a microbe with near 100% certainty, and it would also allow her to identify species with only a few individuals in any sample. Woyke saw a chance to make her mark with these rare but environmentally important species.

    Soon after that, she read Lasken’s paper and decided to try his technique on microbes she had isolated from the grass sharpshooter Draeculacephala minerva, an important plant pest. One of her biggest challenges was contamination. Pieces of DNA are everywhere—on our hands, on tables and lab benches, and in the water. The short, random primers upon which single-cell sequencing was built could help amplify these fragments of DNA just as easily as they could the microbial genomes Woyke was studying. “If someone in the lab had a cat, it could pick up cat DNA,” Woyke says of the technique.

    In 2010, after more than a year of work, Woyke had her first genome, that of Sulcia bacteria, which had a small genome and could only live inside the grass sharpshooter. Each cell also carried two copies of the genome, which helped make Woyke’s work easier. It was a test case that proved the method, but to shine a spotlight on the world’s hidden microbial biodiversity, Woyke would need to figure out how to sequence the genomes from multiple individual microbes.

    Work with Jonathan Eisen, a microbiologist at UC Davis, on the Genomic Encyclopedia of Bacteria and Archaea Project, known as GEBA, enabled her lab to set up a pipeline to perform single cell sequencing on multiple organisms at once. GEBA, which seeks to sequence thousands of bacterial and archaeal genomes, provided a perfect entry to her Microbial Dark Matter sequencing project. More than half of all known bacterial phyla—the taxonomic rank just below kingdom—were only represented by a single 16S rRNA sequence.

    “We knew that there were far more microbes and a far greater diversity of life than just those organisms being studied in the lab,” says Matthew Kane, a program director at the National Science Foundation and a former microbiologist. Studying the select few organisms that scientists could grow in pure culture was “useful for picking apart how cells work, but not for understanding life on Earth.”

    GEBA was a start, but even the best encyclopedia is no match for even the smallest public library. Woyke’s Microbial Dark Matter project would lay the foundation for the first of those libraries. She didn’t want to fill it with just any sequences, however. Common bacteria like E. coli, Salmonella, and Clostridium were the Dr. Seuss books and Shakespeare plays of the microbial world—every library had copies, though they represented only a tiny slice of all published works. Woyke was after the bacterial and archaeal equivalents of rare, single-edition books. So she began searching in extreme environments including boiling hot springs of caustic acid, volcanic vents at the bottom of the ocean, and deep inside abandoned mines.

    Using the single-celled sequencing techniques that she had perfected at the Joint Genome Institute, Woyke and her colleagues ended up with exactly 201 genomes from these candidate phyla, representing 29 branches on the tree of life that scientists knew nothing about. “For many phyla, this was the first genomic data anyone had seen,” she says.

    The results, published in Nature in 2013, identified some unusual species for which even Woyke wasn’t prepared. Up until that study, all organisms used the same sequence of three DNA nucleotides to signal the stop of a protein, one of the most fundamental components of any organism’s genome. Several of the species of archaea identified by Woyke and her colleagues, however, used a completely different stop signal. The discovery was not unlike traveling to a different country and having the familiar red stop sign replaced by a purple square, she says. Their work also identified other rare and bizarre features of the organisms’ metabolisms that make them unique among Earth’s biodiversity. Other microbial dark matter sequencing projects, both under Woyke’s Microbial Dark Matter project umbrella and other independent ventures, identified microbes from unusual phyla living in our mouths.

    Some of the extremeophile archaea that Woyke and her colleagues identified were so unlike other forms of life that they grouped them into their own superset of phyla, known as DPANN (Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanohaloarchaeota, and Nanoarchaeota). The only thing that scientists knew about these organisms were the genomes that Woyke had sequenced, isolated from individual organisms. These single-cell sequencing projects are key not just for filling in the foliage on the tree of life, but also for demonstrating just how much remains unknown, and Woyke and her team have been at the forefront of these discoveries, Kane says.

    Sequencing microbes cell by cell, however, isn’t the only method for uncovering Earth’s hidden biodiversity. Just a few miles from Woyke’s lab, microbiologist Jill Banfield at UC Berkeley is taking a different approach that also has also produced promising results.

    Studying the Uncultured

    Typically, to study microbes, scientists have grown them in a pure culture from a single individual. Though useful for studying these organisms in the laboratory, most microbes live in complex communities of many individuals from different species. Starting in the early 2000s, genetic sequencing technologies had advanced to the point where researchers could study the complex array of microbial genomes without necessarily needing to culture each individual organism. Known as metagenomics, the field began with scientists focused on which genes were found in the wild, which would hint at how each species or strain of microbe could survive in different environments.

    Just as Woyke was doubling down on single-cell sequencing, Banfield began using metagenomics to obtain a more nuanced and detailed picture of microbial ecology. The problems she faced, though very different from Woyke’s, were no less vexing. Like Woyke, Banfield focused on extreme environments: acrid hydrothermal vents at the bottom of the ocean that belched a vile mixture of sulfuric acid and smoke; an aquifer flowing through toxic mine tailings in Rifle, Colorado; a salt flat in Chile’s perpetually parched Atacama Desert; and water found in the Iron Mountain Mine in Northern California that is some of the most acidic found anywhere on Earth. Also like Woyke, Banfield knew that identifying the full range of microbes living in these hellish environments would mean moving away from using the standard set of 16S rRNA primers. The main issue Banfield and colleagues faced was figuring out how to assemble the mixture of genetic material they isolated from their samples into discrete genomes.

    2
    A web of connectivity calculated by Banfield and her collaborators shows how different proteins illustrate relationships between different microbes.
    Credit below.

    The solution wasn’t a new laboratory technique, but a different way of processing the data. Researchers obtain their metagenomic information by drawing a sample from a particular environment, isolating the DNA, and sequencing it. The process of sequencing breaks each genome down into smaller chunks of DNA that computers then reassemble. Reassembling a single genome isn’t unlike assembling a jigsaw puzzle, says Laura Hug, a microbiologist at the University of Waterloo in Ontario, Canada, and a former postdoc in Banfield’s lab.

    When faced with just one puzzle, people generally work out a strategy, like assembling all the corners and edges, grouping the remaining pieces into different colors, and slowly putting it all together. It’s a challenging task with a single genome, but it’s even more difficult in metagenomics. “In metagenomics, you can have hundreds or even thousands of puzzles, many of them might be all blue, and you have no idea what the final picture looks like. The computers have to figure out which blue pieces go together and try to extract a full, accurate puzzle from this jumble,” Hug says. Not surprisingly, the early days of metagenomics were filled with incomplete and misassembled genomes.

    Banfield’s breakthrough helped tame the task. She and her team developed a better method for binning, the formal name for the computer process that sorts through the pile of DNA jigsaw pieces and arranges them into a final product. As her lab made improvements, they were able to survey an increasing range of environments looking for rare and bizarre microbes. Progress was rapid. In the 1980s, most of the bacteria and archaea that scientists knew about fit into 12 major phyla. By 2014, scientists had increased that number to more than 50. But in a single 2015 Nature paper, Banfield and her colleagues added an additional 35 phyla of bacteria to the tree of life.

    4
    The latest tree of life was produced when Banfield and her colleagues added another 35 major groups, known as phyla. Credit below.

    Because researchers knew essentially nothing about these bacteria, they dubbed them the “candidate phyla radiation”—or CPR—the bacterial equivalent of Woyke’s DPANN. Like the archaea, these bacteria were grouped together because of their similarities to each other and their stark differences to other bacteria. Banfield and colleagues estimated that the CPR organisms may encompass more than 15% of all bacterial species.

    “This wasn’t like discovering a new species of mammal,” Hug says. “It was like discovering that mammals existed at all, and that they’re all around us and we didn’t know it.”

    Nine months later, in April 2016, Hug, Banfield, and their colleagues used past studies to construct a new tree of life. Their result reaffirmed Woese’s original 1978 tree, showing humans and, indeed, most plants and animals, as mere twigs. This new tree, however, was much fuller, with far more branches and twigs and a richer array of foliage. Thanks in no small part to the efforts of Banfield and Woyke, our understanding of life is, perhaps, no longer a newborn sapling, but a rapidly maturing young tree on its way to becoming a fully rooted adult.

    Photo credits: Miller et al. 2013/PLOS, Podell et al. 2013/PLOS, Hug et al. 2016/UC Berkeley

    See the full article here .

    Please help promote STEM in your local schools.

    STEM Icon

    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 7:33 am on July 20, 2016 Permalink | Reply
    Tags: , DNA, ,   

    From Princeton: “Role for enhancers in bursts of gene activity (Cell)” 

    Princeton University
    Princeton University

    July 19, 2016
    Marisa Sanders for the Office of the Dean for Research

    A new study by researchers at Princeton University suggests that sporadic bursts of gene activity may be important features of genetic regulation rather than just occasional mishaps. The researchers found that snippets of DNA called enhancers can boost the frequency of bursts, suggesting that these bursts play a role in gene control.

    The researchers analyzed videos of Drosophila fly embryos undergoing DNA transcription, the first step in the activation of genes to make proteins. In a study published on July 14 in the journal Cell, the researchers found that placing enhancers in different positions relative to their target genes resulted in dramatic changes in the frequency of the bursts.

    “The importance of transcriptional bursts is controversial,” said Michael Levine, Princeton’s Anthony B. Evnin ’62 Professor in Genomics and director of the Lewis-Sigler Institute for Integrative Genomics. “While our study doesn’t prove that all genes undergo transcriptional bursting, we did find that every gene we looked at showed bursting, and these are the critical genes that define what the embryo is going to become. If we see bursting here, the odds are we are going to see it elsewhere.”

    The transcription of DNA occurs when an enzyme known as RNA polymerase converts the DNA code into a corresponding RNA code, which is later translated into a protein. Researchers were puzzled to find about ten years ago that transcription can be sporadic and variable rather than smooth and continuous.

    In the current study, Takashi Fukaya, a postdoctoral research fellow, and Bomyi Lim, a postdoctoral research associate, both working with Levine, explored the role of enhancers on transcriptional bursting. Enhancers are recognized by DNA-binding proteins to augment or diminish transcription rates, but the exact mechanisms are poorly understood.

    Until recently, visualizing transcription in living embryos was impossible due to limits in the sensitivity and resolution of light microscopes. A new method developed three years ago has now made that possible. The technique, developed by two separate research groups, one at Princeton led by Thomas Gregor, associate professor of physics and the Lewis-Sigler Institute for Integrative Genomics, and the other led by Nathalie Dostatni at the Curie Institute in Paris, involves placing fluorescent tags on RNA molecules to make them visible under the microscope.

    The researchers used this live-imaging technique to study fly embryos at a key stage in their development, approximately two hours after the onset of embryonic life where the genes undergo fast and furious transcription for about one hour. During this period, the researchers observed a significant ramping up of bursting, in which the RNA polymerase enzymes cranked out a newly transcribed segment of RNA every 10 or 15 seconds over a period of perhaps 4 or 5 minutes per burst. The genes then relaxed for a few minutes, followed by another episode of bursting.

    The team then looked at whether the location of the enhancer – either upstream from the gene or downstream – influenced the amount of bursting. In two different experiments, Fukaya placed the enhancer either upstream of the gene’s promoter, or downstream of the gene and saw that the different enhancer positions resulted in distinct responses. When the researchers positioned the enhancer downstream of the gene, they observed periodic bursts of transcription. However when they positioned the enhancer upstream of the gene, the researchers saw some fluctuations but no discrete bursts. They found that the closer the enhancer is to the promoter, the more frequent the bursting.

    To confirm their observations, Lim applied further data analysis methods to tally the amount of bursting that they saw in the videos. The team found that the frequency of the bursts was related to the strength of the enhancer in upregulating gene expression. Strong enhancers produced more bursts than weak enhancers. The team also showed that inserting a segment of DNA called an insulator reduced the number of bursts and dampened gene expression.

    In a second series of experiments, Fukaya showed that a single enhancer can activate simultaneously two genes that are located some distance apart on the genome and have separate promoters. It was originally thought that such an enhancer would facilitate bursting at one promoter at a time—that is, it would arrive at a promoter, linger, produce a burst, and come off. Then, it would randomly select one of the two genes for another round of bursting. However, what was instead observed was bursting occurring simultaneously at both genes.

    “We were surprised by this result,” Levine said. “Back to the drawing board! This means that traditional models for enhancer-promoter looping interactions are just not quite correct,” Levine said. “It may be that the promoters can move to the enhancer due to the formation of chromosomal loops. That is the next area to explore in the future.”

    The study was funded by grants from the National Institutes of Health (U01EB021239 and GM46638).

    Access the paper here:

    Takashi Fukaya, Bomyi Lim & Michael Levine. Enhancer Control of Transcriptional Bursting, Cell (2016), Published July 14. EPub ahead of print June 9. http://dx.doi.org/10.1016/j.cell.2016.05.025

    See the full article here .

    Please help promote STEM in your local schools.

    STEM Icon

    Stem Education Coalition
    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.

    Princeton Shield

     
  • richardmitnick 12:09 pm on June 8, 2016 Permalink | Reply
    Tags: , DNA, , Second layer of information in DNA confirmed   

    From phys.org: “Second layer of information in DNA confirmed” 

    physdotorg
    phys.org

    June 8, 2016
    Erik Arends

    1
    The rigid base-pair model is forced, using 28 constraints (indicated by red spheres), into a lefthanded superhelical path that mimics the DNA conformation in the nucleosome. Credit: Leiden Institute of Physics

    Leiden theoretical physicists have proven that DNA mechanics, in addition to genetic information in DNA, determines who we are. Helmut Schiessel and his group simulated many DNA sequences and found a correlation between mechanical cues and the way DNA is folded. They have published their results in PLoS One.

    When James Watson and Francis Crick identified the structure of DNA molecules in 1953, they revealed that DNA information determines who we are. The sequence of the letters G, A, T and C in the famous double helix determines what proteins are made ny our cells. If you have brown eyes, for example, this is because a series of letters in your DNA encodes for proteins that build brown eyes. Each cell contains the exact same letter sequence, and yet every organ behaves differently. How is this possible?

    Mechanical cues

    Since the mid 1980s, it has been hypothesized that there is a second layer of information on top of the genetic code consisting of DNA mechanical properties. Each of our cells contains two meters of DNA molecules, and these molecules need to be wrapped up tightly to fit inside a single cell. The way in which DNA is folded determines how the letters are read out, and therefore which proteins are actually made. In each organ, only relevant parts of the genetic information are read. The theory suggests that mechanical cues within the DNA structures determine how preferentially DNA folds.

    Simulation

    For the first time, Leiden physicist Helmut Schiessel and his research group provide strong evidence that this second layer of information indeed exists. With their computer code, they have simulated the folding of DNA strands with randomly assigned mechanical cues. It turns out that these cues indeed determine how the DNA molecule is folded into so-called nucleosomes. Schiessel found correlations between the mechanics and the actual folding structure in the genome of two organisms—baker’s yeast and fission yeast. This finding reveals evolutionary changes in DNA—mutations—that have two very different effects: The letter sequence encoding for a specific protein can change, or the mechanics of the DNA structure can change, resulting in different packaging and levels of DNA accessibility, and therefore differing frequency of production of that protein.

    See the full article here .

    Please help promote STEM in your local schools.

    STEM Icon

    Stem Education Coalition

    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 8:49 am on April 9, 2016 Permalink | Reply
    Tags: , , DNA,   

    From AAAS: “Virus fighter may have played a key role in human evolution” 

    AAAS

    AAAS

    Apr. 7, 2016
    Elizabeth Pennisi

    1
    Some of the body’s antiviral proteins can mess with our DNA—for better or worse. iStock

    A virus-fighting protein in humans and other primates triggers an explosion in genetic mutations that may have sped up the evolution of our species, according to a new study.

    “In some sense, this is scary,” says Kelley Harris, a geneticist at Stanford University in Palo Alto, California, who was not involved with the work. Random mutations are often harmful. But there could be a silver lining: These changes also “provide raw material for evolution to happen” and that may enable individuals besieged by viruses to come up with better antiviral defenses, she says. “The paper doesn’t prove that it’s beneficial for humans to mutate their own DNA when they are infected by viruses, but it’s an interesting possibility.”

    Since the beginning of time, viruses have been inserting their genetic material into the genomes of their hosts, tricking the cell’s machinery into making more virus. Today, our genomes are riddled with these interlopers, called retroviruses and transposable elements, but many now just sit there, unable to generate additional copies of themselves. That’s because our bodies have a group of proteins that have mutated this DNA. These so-called APOBEC proteins seek out certain combinations of the letters that make up DNA (called bases), and, in DNA of viral origin, chemically convert the base cytosine into the base uracil—a change in the genetic alphabet from C to U that can disrupt a gene.

    In 2012, researchers discovered that certain APOBEC proteins do the same in some cancer cells. “You can see they are very active and affect the DNA in the tumor tremendously, causing lots of mutations that may further the cells’ uncontrolled growth, says Alon Keinan, a computational biologist at Cornell University. Because those cancer cells are part of the lungs, kidney, liver, or other organs, the mutations only affect those tissues. But if an APOBEC protein was active in germline cells—those destined to become eggs and sperm—then these mutations could possibly affect future generations, and ultimately alter the course of evolution.

    To see whether this has been the case with one APOBEC protein, APOBEC3G, Erez Levanon, a computational biologist at Bar-Ilan University in Ramat Gan, Israel, contacted Keinan, whose team specializes in comparing genomes to discern patterns of evolution. The group matched the genomes of a modern human, a Denisovan, a Neandertal, and a chimp up against genomes of a mouse, a rhesus macaque, and an orangutan to look for places in the human and chimp genomes with an unusual concentration of changes from a cytosine to another base. They focused only on changes along stretches of DNA with the APOBEC3G protein’s favorite sequence targets. For example, one such favorite is a series of three Cs in a row; the APOBEC3G protein frequently swaps out the third C for a different base.

    All together, the researchers found about 37,000 mutations* occurring in 10,000 clusters in the chimp and human genomes that they think were caused by these proteins, they report today in Genome Research. These mutations were Cs in the orangutan, macaque, and mouse, but a different base in each of the other four species. Many of the clusters were located in key places in the genome, such as regions important for regulating gene activity or protein-coding parts of genes. For example, more than 33% of the base changes they found in coding regions also alter its protein product. Many other changes likely happened in the germlines during the evolution of these species, detrimental ones likely disappeared, whereas those that provided some survival benefit persisted. “It shows that this primate-specific antiviral mechanism also led to the shaping of our and our relatives’ genomes,” Keinan says.

    “It’s surprising to see this impact on all these primate genomes,” says Jeffrey Kidd, a geneticist at the University of Michigan, Ann Arbor, who was not involved with the work. “It makes us realize that nothing comes for free,” and the trade-off of having a mechanism to thwart viral DNA is disruptions in our own DNA, he says. “It raises the question of how that balance is worked out.”

    There are related proteins that may likewise cause mutations; “this might just be the tip of the iceberg,” Keinan says. He and his colleagues are now calculating what percentage of the genetic changes that made us human were caused by APOBEC proteins. Typically, a newborn is expected to have 70 new mutations in its genome, but just one of these proteins “can introduce potentially thousands [of them]” in close proximity and in one generation, Keinan says.

    And that’s a lot of new material for evolution to work on.

    Science Paper:
    Clustered mutations in hominid genome evolution are consistent with APOBEC3G enzymatic activity

    Science team:
    Yishay Pinto1,3; Orshay Gabay 1,3; Leonardo Arbiza 2; Aaron J. Sams 2; Alon Keinan 2,4; and Erez Y. Levanon 1,4

    Author Affiliations

    1 Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 5290002, Israel;
    2 Department of Biological Statistics and Computational Biology, Cornell University, Ithaca, New York 14853, USA

    3,4 not reported

    Corresponding authors: erez.levanon@biu.ac.il, alon.keinan@cornell.edu

    See the full article here .

    The American Association for the Advancement of Science is an international non-profit organization dedicated to advancing science for the benefit of all people.

    Please help promote STEM in your local schools.
    STEM Icon
    Stem Education Coalition

     
  • richardmitnick 5:16 pm on March 23, 2016 Permalink | Reply
    Tags: , DNA, ,   

    From LBL: “Unlocking the Secrets of Gene Expression” 

    Berkeley Logo

    Berkeley Lab

    March 23, 2016
    Julie Chao (510) 486-6491
    JHChao@lbl.gov

    Your DNA governs more than just what color your eyes are and whether you can curl your tongue. Your genes contain instructions for making all your proteins, which your cells constantly need to keep you alive. But some key aspects of how that process works at the molecular level have been a bit of a mystery—until now.

    Using cryo-electron microscopy (cryo-EM), Lawrence Berkeley National Laboratory (Berkeley Lab) scientist Eva Nogales and her team have made a significant breakthrough in our understanding of how our molecular machinery finds the right DNA to copy, showing with unprecedented detail the role of a powerhouse transcription factor known as TFIID.

    1
    Berkeley Lab scientists Eva Nogales and Robert Louder at the electron microscope. (Credit: Roy Kaltschmidt/Berkeley Lab)

    This finding is important as it paves the way for scientists to understand and treat a host of malignancies. “Understanding this regulatory process in the cell is the only way to manipulate it or fix it when it goes bad,” said Nogales. “Gene expression is at the heart of many essential biological processes, from embryonic development to cancer. One day we’ll be able to manipulate these fundamental mechanisms, either to correct for expression of genes that should or should not be present or to take care of malignant states where the process has gone out of control.”

    Their study has been published online in the journal Nature in an article titled, Structure of promoter-bound TFIID and insight into human PIC assembly. The lead author is Robert Louder, a biophysics graduate student in Nogales’ lab, and other authors are Yuan He, José Ramón López-Blanco, Jie Fang, and Pablo Chacón.

    Nogales, a biophysicist who also has appointments at Howard Hughes Medical Institute and UC Berkeley, has been studying gene expression for 18 years. While she and her team have made several significant findings in recent years, she calls this the biggest breakthrough so far. “This is something that will go in biochemistry textbooks,” she said. “We now have the structure of the whole protein organization that is formed at the beginning of every gene. This is something no one has come close to doing because it is really very difficult to study by traditional methodologies.”

    2
    Cryo-EM model of the human transcription pre-initiation complex. (Credit: Robert Louder/Berkeley Lab )

    How genetic information flows in living organisms is referred to as the “central dogma of molecular biology.” Cells are constantly turning genes on and off in response to what’s happening in their environment, and to do that, the cell uses its DNA, the big library of genetic blueprints, finds the correct section, and makes a copy in the form of messenger RNA; the mRNA is then used to produce the needed protein.

    The problem with this “library” is that it has no page numbers or table of contents. However, markers are present in the form of specific DNA sequences (called core promoter motifs) to indicate where a gene starts and ends. So how does the polymerase, the enzyme that carries out the transcription, know where to start? “DNA is a huge, huge molecule. Out of this soup, you have to find where this gene starts, so the polymerase knows where to start copying,” Nogales said. “This transcription factor, TFIID, is the protein complex that does exactly that, by recognizing and binding to DNA core promoter regions.”

    What Nogales and her team have been able to do is to visualize, with unprecedented detail, TFIID bound to DNA as it recognizes the start, or promoter, region of a gene. They have also found how it serves as a sort of landing pad for all the molecular machinery that needs to assemble at this position—this is called the transcription pre-initiation complex (PIC). This PIC ultimately positions the polymerase so it can start transcribing.

    3
    TFIID (blue) as it contacts the DNA and recruits the polymerase (grey) for gene transcription. The start of the gene is shown with a flash of light. (Credit: Eva Nogales/Berkeley Lab)

    “TFIID has to do not only the binding of the DNA, recruitment, and serving as landing pad, it has to somehow do all that differently for different genes at any given point in the life of the organism,” Nogales said.

    Added Louder: “We have generated the first ever structural model of the full human TFIID-based PIC. Our model yields novel insights into human PIC assembly, including the role of TFIID in recruiting other components of the PIC to the promoter DNA and how the long observed conformational flexibility of TFIID plays a role in the regulation of transcription initiation.”

    Proteins have traditionally been studied using X-ray crystallography, but that technique has not been possible for this kind of research. “TFIID has not been accessible to protein crystallography because there’s not enough material to crystallize it, it has very flexible elements, and it is of a huge size,” Nogales said. “All of those things we can overcome through cryo-EM.”

    Cryo-EM, in which samples are imaged at cryogenic temperatures without need for dyes or fixatives, has been used since the 1980s in structural biology. With extensive computational analysis of the images researchers are able to obtain three-dimensional structures. However, cryo-EM has undergone a revolution in the last few years with the advent of new detectors—developed, in fact, at Berkeley Lab—that improve resolution and reduce the amount of data needed by up to a hundred-fold.

    “Many biological systems we had thought were impossible to study at high resolution have become accessible,” she said. “Now the resolution allows us to get atomic details. This is an area in which Berkeley Lab has been one of the leaders.”

    While this study has revealed important new insights into gene expression, Nogales notes that much work remains to be done. Next she plans to investigate how TFIID is able to recognize different sequences for different gene types and also how it is regulated by cofactors and activators.

    “We are just at the beginning,” she said. “This complex, TFIID, is very, very critical. Now we have broken barriers in the sense that we can start generating atomic models and get into details of how DNA is being bound.”

    This research was supported by the National Institutes of Health’s National Institute of General Medical Sciences and by the Spanish Ministry of Economy and Competitiveness. Computational work was carried out at the National Energy Research Scientific Computing Center (NERSC), a DOE Office of Science User Facility hosted at Berkeley Lab. Nogales is a Senior Faculty Scientist in Berkeley Lab’s Molecular Biophysics and Integrated Bioimaging Division; additional information on her lab can be found here.

    See the full article here .

    Please help promote STEM in your local schools.

    STEM Icon

    Stem Education Coalition

    A U.S. Department of Energy National Laboratory Operated by the University of California

    University of California Seal

    DOE Seal

     
  • richardmitnick 6:50 am on February 6, 2016 Permalink | Reply
    Tags: , , DNA,   

    From SPIE: “Tracking DNA damage with electrochemical sensing” 

    SPIE

    SPIE

    2.6.16
    Jason D. Slinker
    The University of Texas at Dallas
    Richardson, TX

    DNA, the fundamental biomolecule of life, is constantly subject to damage that threatens the vitality of cells and the integrity of the genome. Without enzymatic intervention, this damage can produce mutations that lead to cancerous tumors. Furthermore, many current and developing treatments of cancer and disease rely on the generation of DNA damage products, which—from a chemical standpoint—are very subtle. For example, 8-oxoguanine, the most prevalent oxidative DNA damage product, involves the addition of a single oxygen bond to a guanine base. Remarkably, enzymes in cells recognize and remove this damage and other products of degradation. Biological assays that follow repair of this subtle DNA damage assist cancer studies by advancing fundamental understanding of DNA-protein interactions, connecting damage to diagnosis, and informing options for treatment.

    We have demonstrated devices that follow DNA damage repair in real time, with a convenient, low-cost package (see Figure 1).1 In this device, DNA is bound to the circular electrodes of multielectrode chips, and a redox probe at the top of the DNA reports charge transfer through it. DNA is the natural recognition element not only for the binding of repair proteins but also for their repair activity, and it can be synthesized with or without damage/lesion sites to establish controls. Furthermore, DNA can also serve as an electrical transducing element when modified with a redox-active probe and self-assembled on a working electrode, as first demonstrated by the Barton group.2 We have combined these features of DNA, using them to form devices capable of selectively detecting oxidative DNA damage repair (see Figure 1) and changes in DNA stability.1 The devices give a direct measure of molecular-level repair, providing a window into intracellular DNA repair by DNA-binding proteins.

    DNA Device
    Figure 1. Top: Schematic of detection of oxidative damage removal. Bottom: Image of the device used to study DNA-damaging drugs. (Photo by Randy Anderson). FPG: Formamidopyrimidine DNA glycosylase. e-: Electron.

    Specifically, we have used our approach to show sensitive and selective electrochemical sensing of 8-oxoguanine and uracil repair glycosylase activity. We produced sensors on electrospun fibers as low-cost devices with improved dynamic range. Our experiments compared electroactive, probe-modified DNA monolayers containing a base defect with the rational control of defect-free monolayers. We found damage-specific limits of detection on the order of femtomoles of proteins, corresponding to mere nanograms of the enzymes. The DNA chips enabled the real-time observation of protein activity, and we observed base excision activity on the order of seconds. We also demonstrated damage-specific detection in a mixture of enzymes and in response to environmental oxidative damage. We showed how nanofibers may behave similarly to conventional gold-on-silicon devices, revealing the potential of these low-cost devices for sensing applications. This device approach enables sensitive, selective, and rapid assay of repair protein activity, allowing biological interrogation of DNA damage repair.

    Given the ability of these devices to follow induced oxidative damage, we are further using them to follow DNA-damaging anticancer drug activity. We are working with the group of David Boothman of the University of Texas Southwestern Medical Center to sense DNA repair activity in conjunction with a novel drug therapy that selectively produces oxidative damage of DNA in cancer cells, bringing about selective cancer cell death. We represent key features of a living system to reproduce DNA damaging and repair activity pathways on the chip. Recent results have shown that we can follow specific drug-induced DNA damage excision and subsequent DNA repair with our devices. Furthermore, the multiple electrodes of the chip allowed us to perform controls of each associated enzyme and to obtain high statistical confidence of results. Given this success, we have launched studies of other DNA damaging drugs to explore the generality of this technique.

    In summary, we have designed and fabricated low-cost devices that are capable of electrochemical sensing of 8-oxoguanine and uracil repair glycosylase activity. Ultimately, in addition to their utility in bioassays of DNA-protein interactions, our devices have potential in a number of applications for public health, and our future work will focus on realizing these. The prevalence of high damage repair sites can be an indication of cancers and disease states, and these devices could provide statistically significant diagnosis. Additionally, as a number of cancer treatments involve DNA-damaging agents, our devices can be used to improve treatment outcomes. These devices could be used to sample the activity of multiple drugs with a small volume patient sample, enabling a tailored treatment based on DNA-damaging effectiveness. Similarly, they may also be used to follow the course of cancer treatment through characteristic measures of enzymatic activity of cancer cells versus healthy cells.

    See the full article here.

    Please help promote STEM in your local schools.

    STEM Icon

    Stem Education Coalition

     
  • richardmitnick 2:07 am on January 23, 2016 Permalink | Reply
    Tags: , DNA,   

    From Vanderbilt: “Faulty building blocks in DNA” 

    Vanderbilt U Bloc

    Vanderbilt University

    Jan. 22, 2016
    Bill Snyder

    Temp 1
    (iStock)

    Enzymes called DNA polymerases assemble DNA from 2´-deoxyribonucleoside triphosphate building blocks in the cell. Normally they can distinguish DNA building blocks from the ribonucleotides that make up RNA, but sometimes they misinsert ribonucleotides into DNA, generating “DNA lesions.”

    Yan Su, Ph.D., Martin Egli, Ph.D., and F. Peter Guengerich, Ph.D., have provided an important glimpse into how this happens. They studied human DNA polymerase eta (hpol-eta), which is directly related to a human genetic disorder, xeroderma pigmentosum, associated with an increased risk of skin and other cancers.

    In a paper published online this month by the Journal of Biological Chemistry, they show that hpol-eta can incorporate ribonucleotides into DNA with relatively high selectivity but low efficiency. They also crystallized the enzyme and obtained what appears to be the first crystal structure of an incoming ribonucleotide opposite a DNA lesion within a DNA polymerase. Based on these findings, “it is highly possible that hpol-eta inserts a considerable amount of ribonucleotides into DNA,” they conclude.

    This work was supported by National Institutes of Health.

    See the full article here .

    Please help promote STEM in your local schools.

    STEM Icon

    Stem Education Coalition

    Vanderbilt Campus

     
  • richardmitnick 2:56 pm on January 6, 2016 Permalink | Reply
    Tags: , , , DNA   

    From AAAS: “Researchers rein in slice-happy gene editor, CRISPR” 

    AAAS

    AAAS

    6 January 2016
    Kelly Servick

    Temp 1
    Adapted from H. Nishimasu et al., Cell, 156, 5 (2014); Wikimedia/Creative Commons

    Changes to the DNA-cutting enzyme Cas9 make CRISPR more precise.

    Keith Joung remembers the first time he took CRISPR for a spin. In late 2012, the pathologist at Massachusetts General Hospital in Boston assembled the components of the new gene-editing technology and fiddled with the DNA of a zebrafish embryo. “It was so easy to do,” he says. “It was just stunning.”

    CRISPR—the highly efficient set of molecular scissors recently selected as Science’s Breakthrough of the Year—might be easy to use, but it’s not perfect. Joung and his colleagues soon found that these scissors could get too slice-happy, cutting DNA in unexpected and unwanted locations. In early experiments, the group observed that these off-target effects could occur at some DNA sites with nearly the same frequency as the desired edits. That’s a problem if CRISPR is to form the basis of human therapies, for example, repairing the defective genes that cause muscular dystrophy or hereditary liver disease. Researchers’ primary concern is that cutting into an unwanted gene could cause uncontrolled growth and cancer.

    Now, Joung and colleagues have found a way to make CRISPR more precise. In a new study, they modified its cutting enzyme to reduce off-target effects below detectable levels.

    “I think that this is a potential breakthrough,” says Jin-Soo Kim, a molecular biologist at Seoul National University who was not involved with the work. But the quest to perfect CRISPR doesn’t have a clear end. “No drugs are free of off-target effects,” he notes. With CRISPR-based therapies still far from human testing, no one knows just how precise is precise enough.

    CRISPR relies on a DNA-cutting enzyme called Cas9 attached to a short strand of RNA that guides it to specific point in the genome. When the RNA finds a complementary—or nearly complementary—sequence, Cas9 makes its slice. There are already several approaches to prevent unintended slicing. Shortening the length of the guide RNA makes it more sensitive to mismatched sequences, but it can also create entirely new off-target effects. Some labs have experimented with a version of Cas9 that cuts through a single DNA strand instead of two. That means two Cas9 enzymes bearing two different guide RNAs have to recognize their target sequences to cut both strands—a more demanding matching process. But doubling the number of RNA guides adds bulk, which could make it harder to deliver a CRISPR-based treatment into cells.

    In the new work, published online today in Nature, Joung and colleagues took a different approach. They modified the Cas9 enzyme itself to change the way it interacts with DNA. They first altered some of the “residues” on the enzyme’s surface that presumably help the guide RNA pair with its matching DNA strand. One set of modifications created a new variant of Cas9, called Cas9-HF1, that appears to be much more discriminating in its cuts. The researchers made seven different edits guided by seven different RNA strands, each known to produce off-target effects with Cas9. But Cas9-HF1 showed no detectable off-target effects in six of these cases—and just one errant slice in the seventh, they report. Joung adds that the apparent slice could actually be the result of a sequencing error.

    The results come on the heels of a similar feat, led by CRISPR pioneer Feng Zhang of Harvard University and the Broad Institute in Cambridge, Massachusetts, published last month in Science. That team modified Cas9 to change how it interacts with a different part of a cell’s DNA. It, too, dramatically improved CRISPR’s specificity. But it’s hard to compare those results directly with the new paper because they used slightly different methods to measure off-target effects.

    Joung claims his group’s measurements are roughly 10-fold more sensitive than the one used in the Science paper. Both studies rely on methods that attach molecular tags to all points in the genome where a double-stranded break has occurred, before sequencing the short, flagged segments to count the cuts in various genes. Joung’s team claims to detect edits that occur in at least 0.1% of the genome. Zhang says the method used in his paper has been validated down 0.3%, and it may be even more sensitive.

    Does detecting just a couple of faulty cuts in a thousand matter? Absolutely, Joung says. “A lot of therapeutic strategies envision manipulating millions, tens of millions, even hundreds of millions of cells, potentially. So one in 1000 sounds pretty good, but that number can become quite large.” He argues that the field needs tests that root out these potentially harmful effects at frequencies of 0.01% or even lower.

    Others are less focused on increasingly sensitive tests. Because CRISPR will never fully be rid of off-target effects, the key question for a given therapy is not strictly how many unwanted cuts it makes, but whether it disrupts any essential genes, says Jiing-Kuan Yee, a molecular biologist at the research center City of Hope in Duarte, California. Each therapeutic application will require its own carefully selected Cas9 molecule—and modifications like those in the two recent papers might be combined.

    “Pretty soon, I think everybody’s going to start using these modified Cas9s,” he says. “The [off-target] problem will still be there, but it’s going to be much, much reduced.”

    See the full article here .

    The American Association for the Advancement of Science is an international non-profit organization dedicated to advancing science for the benefit of all people.

    Please help promote STEM in your local schools.
    STEM Icon
    Stem Education Coalition

     
  • richardmitnick 5:14 pm on December 28, 2015 Permalink | Reply
    Tags: , DNA, ,   

    From NOVA: “The Man Who Rewrote the Tree of Life” 2014 but Interesting and Important 

    PBS NOVA

    NOVA

    30 Apr 2014
    Carrie Arnold

    Carl Woese may be the greatest scientist you’ve never heard of. “Woese is to biology what [Albert] Einstein is to physics,” says Norman Pace, a microbiologist at the University of Colorado, Boulder. A physicist-turned-microbiologist, Woese specialized in the fundamental molecules of life—nucleic acids—but his ambitions were hardly microscopic. He wanted to create a family tree of all life on Earth.

    Woese certainly wasn’t the first person with this ambition. The desire to classify every living thing is ageless. The Ancient Greeks and Romans worked to develop a system of classifying life. The Jewish people, in writing the Book of Genesis, set Adam to the task of naming all the animals in the Garden of Eden. And in the mid-1700s, Swedish botanist Carl von Linné published Systema Naturae, introducing the world to a system of Latin binomials—Genus species—that scientists use to this day.

    Temp 1
    Carl Woese in his later years. Photo credits: Jason Lindsey/University of Illinois, Tim Bocek/Flickr (CC BY-NC-SA)

    What Woese was proposing wasn’t to replace Linnaean classification, but to refine it. During the late 1960s, when Woese first started thinking about this problem as a young professor at the University of Illinois, biologists were relying a lot on guesswork to determine how organisms were related to each other, especially microbes. At the time, researchers used the shapes of microbes—their morphologies—and how they turned food into energy—their metabolisms—to sort them into bins. Woese was underwhelmed. To him, the morphology-metabolism approach was like trying to create a genealogical history using only photographs and drawings. Are people with dimples on their right cheeks and long ring fingers all members of the same family? Maybe, but probably not.

    “If you wanted to build a tree of life prior to what Woese did, there was no way to put something together that was based upon actual data,” says Jonathan Eisen, an evolutionary microbiologist at the University of California Davis.

    Just as outward appearances aren’t the best way to determine family relations, Woese believed that morphology and metabolism were inadequate classifiers for life on Earth. Instead, he figured that DNA could sketch a much more accurate picture. Today, that approach may seem like common sense. But in the late 60s and early 70s, this was no easy task. Gene sequencing was a time-consuming, tedious task. Entire PhDs were granted for sequencing just one gene. To create his tree of life, Woese would need to sequence the same gene in hundreds, if not thousands, of different species.

    So Woese toiled in his lab, sometimes with his postdoc George Fox but often alone, hunched over a light box with a magnifying glass, sequencing genes nucleotide by nucleotide. It took more than a decade. “When Woese first announced his results, I thought he was exaggerating at first,” Fox recalls. “Carl liked to think big, and I thought this was just another of his crazy ideas. But then I looked at the data and the enormity of what we had discovered hit me.”

    Woese and Fox published their results in 1977 in a well-respected journal, the Proceedings of the National Academy of Science. They had essentially rewritten the tree of life. But Woese still had a problem: few scientists believed him. He would spend the rest of his life working to convince the biological community that his work was correct.

    Animal, Vegetable, Mineral

    Following the publication of Linnaeus’s treatise in the 18th century, taxonomy progressed incrementally. The Swedish botanist had originally sorted things into three “kingdoms” of the natural world: animal, vegetable, and mineral. He placed organisms in their appropriate cubbyholes by looking at similarities in appearance. Plants with the same number of pollen-producing stamens were all lumped together, animals with the same number of teeth per jaw were grouped, and so on. With no knowledge of evolution and natural selection, he didn’t have a better way to comprehend the genealogy of life on Earth.

    The publication of [Charles]Darwin’s On the Origin of Species in 1859, combined with advances in microscopy, forced scientists to revise Linnaeus’s original three kingdoms to include the tiniest critters, including newly visible ones like amoebae and E. coli. Scientists wrestled with how to integrate microbial wildlife into the tree of life for the next 100 years. By the mid-20th century, however, biologists and taxonomists had mostly settled on a tree with five major branches: protists, fungi, plants, animals, and bacteria. It’s the classification system that many people learned in high school biology class.

    Woese and other biologists weren’t convinced, though. Originally a physics major at Amherst College in Massachusetts and having received a PhD in biophysics from Yale in 1953, Woese believed that there had to be a more objective, data-driven way to classify life. Woese was particularly interested in how microbes fit into the classification of life, which had escaped a rigorous genealogy up until that point.

    He arrived at the University of Illinois Urbana-Champaign as a microbiologist in the mid-1960s, shortly after James Watson and Francis Crick won the Nobel prize for their characterization of DNA’s double-helix form. It was the heyday of DNA. Woese was enthralled. He believed that DNA could unlock the hidden relationships between different organisms. In 1969, Woese wrote a letter to Crick, stating that:

    ” …this can be done by using the cell’s ‘internal fossil record’—i.e., the primary structures of various genes. Therefore, what I want to do is to determine primary structures for a number of genes in a very diverse group of organisms, on the hope that by deducing rather ancient ancestor sequences for these genes, one will eventually be in the position of being able to see features of the cell’s evolution….”

    This type of thinking was “radically new,” says Norman Pace, a microbiologist at the University of Colorado, Boulder. “No one else was thinking in this direction at the time, to look for sequence-based evidence of life’s diversity.”

    Evolution’s Timekeeper

    Although the field of genetics was still quite young, biologists had already figured out some of the basics of how evolution worked at the molecular level. When a cell copies its DNA before dividing in two, the copies aren’t perfectly identical. Mistakes inevitably creep in. Over time, this can lead to significant changes in the sequence of nucleotides and the proteins they code for. By finding genes with sites that mutate at a known rate—say 4 mutations per site per million years—scientists could use them as an evolutionary clock that would give biologists an idea of how much time had passed since two species last shared a common ancestor.

    To create his evolutionary tree of life, then, Woese would need to choose a gene that was present in every known organism, one that was copied from generation to generation with a high degree of precision and mutated very slowly, so he would be able to track it over billions of years of evolution.

    “This would let him make a direct measure of evolutionary history,” Pace says. “By tracking these gene sequences over time, he could calculate the evolutionary distance between two organisms and make a map of how life on Earth may have evolved.”

    Some of the most ancient genes are those coding for molecules known as ribosomal RNAs. In ribosomes, parts of the cell that float around the soupy cytoplasm, proteins and ribosomal RNA, or rRNA, work together to crank out proteins. Each ribosome is composed of large and small subunits, which are similar in both simple, single-celled prokaryotes and more complex eukaryotes. Woese had several different rRNA molecules to choose from in the various subunits, which are classified based on their length. At around 120 nucleotides long, 5S rRNA wasn’t big enough to use to compare lots of different organisms. On the other end of the spectrum, 23S rRNA was more than 2300 nucleotides long, making it far too difficult for Woese to sequence using the technologies of the time. The Goldilocks molecule—long enough to allow for meaningful comparisons but not too long and difficult to sequence—was 16S rRNA in prokaryotes and its slightly longer eukaryotic equivalent, 18S rRNA. Woese decided to use these to create his quantitative tree of life.

    His choice was especially fortuitous, Eisen says, because of several factors inherent in 16S rRNA that Woese couldn’t have been aware of at the time, including its ability to measure evolutionary time on several different time scales. Certain parts of the 16S rRNA molecule mutate at different speeds. Changes to 16S rRNA are, on the whole, still extremely slow (humans share about 50% of their 16S rRNA sequence with the bacterium E. coli), but one portion mutates much more slowly than the other. It’s as if the 16S rRNA clock has both an hour hand and a minute hand. The very slowly evolving “hour hand” lets biologists study the long-term changes to the molecule, whereas the more quickly evolving “minute hand” provides a more recent history. “This gives this gene an advantage because it lets use ask questions about deep evolutionary history and more recent history at the same time,” Eisen says.

    Letter by letter

    Selecting the gene was just Woese’s first challenge. Now he had to sequence it in a variety of different organisms. In the late 60s and early 70s, when Woese began his work, DNA sequencing was far from automated. Everything, down to the last nucleotide, had to be done by hand. Woese used a method to catalog short pieces of RNA developed in 1965 by British scientist Frederick Sanger, which used enzymes to chop RNA into small pieces. These small pieces were sequenced, and then scientists had to reassemble the overlapping pieces to determine the overall sequence of the entire molecule—a process that was tedious, expensive, and time-consuming, but that was seen as a minor annoyance to a workhorse like Woese, Fox says. “All he cared about was getting the answer.”

    Woese started with prokaryotes, the single-celled organisms that were his primary area of interest. He and his lab started by growing bacteria in a solution of radioactive phosphate, which the cells incorporated into backbones of their RNA molecules. This made the 16S rRNA radioactive. Then, Woese and Fox extracted the RNA from the cells and chopped it into smaller pieces using enzymes that acted like scissors. The enzymatic scissors would only cut at certain sequences. If a sequence was present in one organism but missing in a second, the scissors would pass over the second one’s sequence. Its fragment would be longer.

    Since RNA’s sugar-phosphate backbone is negatively charged, the researchers could use a process known as electrophoresis to separate the different length pieces. As electricity coursed through gels containing samples, it pulled the smaller, lighter bits farther through the gels than the longer, heavier chunks. The result was distinct bands of different lengths of RNA. Woese and Fox then exposed each gel to photographic paper over several days. The radioactive bands in the gel transferred marks to the paper. This created a Piet Mondrian-esque masterpiece of black bands on a white background. Each different organism left its own mark. “To Carl, each spot was a puzzle that he would solve,” Fox says.

    After developing each image, Woese and Fox returned to the gel and neatly cut out each individual blotch that contained fragments of a certain length. They then chopped up these fragments with another set of enzymes until they were about five to 15 nucleotides long, a length that made sequencing easier. For some of the longer fragments, it took several iterations of the process before they were successfully sequenced. The sequences were then recorded on a set of 80-column IBM punch cards. The cards were then run through a large computer to compare band patterns and RNA sequences among different organisms to determine evolutionary relationships. At the beginning, it took Woese and Fox months to obtain a single 16S rRNA fingerprint.

    “This process was a huge breakthrough,” says Peter Moore, an RNA chemist at Yale University who worked with Woese on other research relating to RNA’s structure. “It gave biologists a tool for sorting through microorganisms and giving them a conceptual way to understand the relationship between them. At the time, the field was just a total disaster area. Nobody knew what the hell was going on.”

    RNA is so fundamental to life that some scientists think it’s the spark that started it all. To learn more about RNA, visit NOVA’s RNA Lab.

    By the spring of 1976, Woese and Fox had created fingerprints of a variety of bacterial species when they turned to an oddball group of prokaryotes: methanogens. These microbes produce methane when they break down food for energy. Because even tiny amounts of oxygen are toxic to these prokaryotes, Woese and Fox had to grow them under special conditions.

    After months of trial and error, the two scientists were finally able to obtain an RNA fingerprint of one type of methanogen. When they finally analyzed its fingerprint, however, it looked nothing like any of the other bacteria Woese and Fox had previously analyzed. All of the previous bacterial gels contained two large splotches at the bottom. They were entirely absent from these new gels. Woese knew instantly what this meant.

    To fellow microbiologist Ralph Wolfe, who worked in the lab next door, Woese announced, “I don’t even think these are bacteria, Wolfe.”

    He dropped the full bombshell on Fox. “The methanogens didn’t have any of the spots he was expecting to see. When he realized this wasn’t a mistake, he just went nuts. He ran into my lab and told me we had discovered a new form of life,” Fox recalls.

    The New Kingdom

    The methanogens Woese and Fox had analyzed looked superficially like other bacteria, yet their RNA told a different story, sharing more in common with nucleus-containing eukaryotes than with other bacteria. After more analysis of his RNA data, Woese concluded that what he was tentatively calling Archaea (from Latin, meaning primitive) wasn’t a minor twig on the tree of life, but a new main branch. It wasn’t just Bacteria and Eukarya any more .

    To prove to their critics that these prokaryotes really were a separate domain on the tree of life, Woese and Fox knew the branch needed more than just methanogens. Fox knew enough about methanogen biology to know that their unique RNA fingerprint wasn’t the only thing that made them strange. For one thing, their cell walls lacked a mesh-like outer layer made of peptidoglycan. Nearly every other bacterium Fox could think of contained peptidoglycan in its cell wall—until he recalled a strange fact he had learned as a graduate student—another group of prokaryotes, the salt-loving halophiles, also lacked peptidoglycan.

    2
    Grand Prismatic Spring in Yellowstone National Park is home to many species of thermophilic archaea.

    Fox turned to the research literature to search for other references to prokaryotes that lack peptidoglycan. He found two additional examples: Thermoplasma and Sulfolobus. Other than the missing peptidoglycan, these organisms and the methanogens seemed nothing alike. Methanogens were found everywhere from wetlands to the digestive tracts, halophiles flourished in salt, Thermoplasma liked things really hot, and Sulfolobus are often found in volcanoes and hot, acidic springs.

    Despite their apparent differences, they all metabolized food in the same, unusual way—unlike anything seen in other bacteria—and the fats in the cell membrane were alike, too. When Woese and Fox sequenced the 16S rRNA of these organisms, they found that these prokaryotes were most similar to the methanogens.

    “Once we had the fingerprints, it all fell together,” Fox says.

    Woese believed his findings were going to revolutionize biology, so he organized a press conference when the paper was published in PNAS in 1977. It landed Woese on the front page of the New York Times, and created animosity among many biologists. “The write-ups were ludicrous and the reporters got it all wrong,” Wolfe says. “No biologists wanted anything to do with him.”

    It wasn’t just distaste for what looked like a publicity stunt that was working against Woese. He had spent most of the last decade holed up in his third floor lab, poring over RNA fingerprints. His reclusive nature had given him the reputation of a crank. It also didn’t help that he had single-handedly demoted many biologists’ favorite species. Thanks to Woese, Wolfe says, “Microbes occupy nearly all of the tree. Then you have one branch at the very end where all the animals and plants were. And the biologists just couldn’t believe that all the plants and all the animals were really just one tiny twig on one branch.”

    Although some specialists were quick to adopt Woese’s new scheme, the rest of biology remained openly hostile to the idea. It wasn’t until the mid-1980s that other microbiologists began to warm to the idea, and it took well over another decade for other areas of biology to follow suit. Woese had grown increasingly bitter that so many other scientists were so quick to reject his claims. He knew his research and ideas were solid. But he was left to respond to what seemed like an endless stream of criticism. Shying from these attacks, Woese retreated to his office for the next two decades.

    “He was a brash, iconoclastic outsider, and his message did not go down well,” says Moore, the Yale RNA chemist.

    Woese’s cause wasn’t helped by his inability to engage critics in dialogue and discussion. Both reticent and abrupt, he preferred his lab over conferences and presentations. In place of public appearances to address his detractors, he sent salvos of op-eds and letters to the editor. Still, nothing seemed to help. The task of publicly supporting this new tree of life fell to Woese’s close colleagues, especially Norman Pace.

    But as technology improved, scientists began to obtain the sequences of an increasing number of 16S rRNAs from different organisms. More and more of their analyses supported Woese’s hypothesis. As sequencing data poured in from around the world, it became clear to nearly everyone in biology that Woese’s initial tree was, in fact, been correct.

    Now, when scientists try to discover unknown microbial species, the first gene they sequence is 16S rRNA. “It’s become one of the fundamentals of biology,” Wolfe says. “After more than 20 years, Woese was finally vindicated.”

    Woese died on December 30, 2012, at the age of 84 of complications from pancreatic cancer. At the time of his death, he had won some of biology’s most prestigious awards and had become one of the field’s most respected scientists. Thanks to Woese’s legacy, we now know that most of the world’s biodiversity is hidden from view, among the tiny microbes that live unseen in and around us, and in them, the story of how life first evolved on this planet.

    See the full article here .

    Please help promote STEM in your local schools.

    STEM Icon

    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.

     
c
Compose new post
j
Next post/Next comment
k
Previous post/Previous comment
r
Reply
e
Edit
o
Show/Hide comments
t
Go to top
l
Go to login
h
Show/Hide help
shift + esc
Cancel
%d bloggers like this: