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  • richardmitnick 8:50 am on September 1, 2014 Permalink | Reply
    Tags: , , , DNA, RNA   

    From Astrobio: “DNA May Have Had Humble Beginnings As Nutrient Carrier” 

    Astrobiology Magazine

    Astrobiology Magazine

    Sep 1, 2014
    Adam Hadhazy

    New research intriguingly suggests that DNA, the genetic information carrier for humans and other complex life, might have had a rather humbler origin. In some microbes, a study shows, DNA pulls double duty as a storage site for phosphate. This all-important biomolecule contains phosphorus, a sometimes hard-to-get nutrient.

    dna

    Maintaining an in-house source of phosphate is a newfound tactic for enabling microorganisms to eke out a living in harsh environments, according to a new study published in the open-access, peer reviewed scientific journal PLOS ONE. The finding bodes well for life finding a way, as it were, in extreme conditions on worlds less hospitable than Earth.

    The results also support a second insight: DNA might have come onto the biological scene merely as a means of keeping phosphate handy. Only later on in evolutionary history did the mighty molecule perhaps take on the more advanced role of genetic carrier.

    “DNA might have initially evolved for the purpose of storing phosphate, and the various genetic benefits evolved later,” said Joerg Soppa, senior author of the paper and a molecular biologist at Goethe University in Frankfurt, Germany.

    Unraveling life’s origins

    Scientists continue to investigate the development of self-replicating, intricate sets of chemistry — in other words, life — from the chemical compounds thought available on early Earth. Out of this mixture of prebiotic chemicals, two nucleic acids — RNA and DNA — emerged as champions.

    early
    Early Earth, in an artist’s impression, where somehow complex, self-replicating chemistry (in other words, life) emerged. Credit: Peter Sawyer / Smithsonian Institution

    Today, these two types of biomolecules serve as the genetic information carriers for all Earthly biota. RNA on its own suffices for the business of life for simpler creatures, such as some viruses. Complex life, like humans, however, relies on DNA as its genetic carrier.

    Astrobiologists want to understand the origin of DNA and its genetic cousin, RNA, because figuring out how life got started here on Earth is key for gauging if it might ever develop on alien planets.

    Many researchers think RNA must have preceded DNA as the genetic molecule of choice. RNA is more versatile, acting as both genetic code and a catalyst for chemical reactions. Explicating the rise of DNA as a genetic material directly from RNA, however, is tricky. Compared to RNA, DNA needs significantly more supporting players for it to work well in a biological setting.

    “The switch from RNA to DNA is not easy because many additional enzymes are required for DNA genomes,” said Soppa.

    This unclear transition from RNA to DNA opens the door for a precursor to DNA possibly having a more mundane job. The new study offers an attractive explanation: that DNA was a fancy way to store nutrients in cells.

    Phosphate depot?

    DNA is chock-full of phosphate. Cells depend on phosphate to form not only DNA and RNA, but also related genetic machinery, such as the ribosome. Phosphate, furthermore, is a must for building the molecule ATP, life’s energy carrier, as well as fatty membrane molecules, certain phospho-proteins and phospho-sugars, and more.

    ds
    The shores of the Dead Sea, which borders Jordan, Palestine and Israel. As the lowest and saltiest lake in the world, it is home to some extreme creatures. Image Credit: Aaron L. Gronstal

    “Phosphate is important for an immense set of biomolecules,” said Soppa.

    Unfortunately for some microbes, ample phosphate is not always available. For example, in salty, nutrient-poor habitats, such as the Dead Sea in the Middle East, an organism called Haloferax volcanii must regularly “eat” ambient DNA to obtain phosphate (plus some other nutritional goodies, such as nitrogen).

    Notably, H. volcanii can still survive and reproduce when phosphorus, the element needed to make phosphate, is lacking. Somehow, then, the microbe must turn to an inner source of phosphate, for otherwise it should cease to grow.

    In their study, Soppa and colleagues from Germany, the United States and Israel sought out this source. The nature of H. volcanii provided some clues. The organism is classified as archaea, one of the three domains of life, in addition to bacteria and eukarya, the latter encompassing all multicellular organisms, from fungi to fruit flies. Many archaea and bacteria — collectively, “prokaryotes”— have just one, circular chromosome. Eukaryotes, like us, on the other hand, can have any number of the chunky pieces of DNA, RNA and proteins. (Humans have 23 pairs of different chromosomes, for the record.) H. volcanii is unusual. It has 20 copies of the same chromosome when it’s growing happily under favorable conditions, and 10 when nutrients are exhausted and it reaches a stationary phase.

    Strength in numbers

    Lots of chromosome copies are good to have in a pinch. So-called polyploidal organisms like H. volcanii use their copious chromosomes to tough it out through bad situations, such as high radiation exposure or total dry-outs, called desiccation. Either scenario causes the strands in chromosomal DNA to break. For single-chromosome species, only a few breaks lead to death because it is impossible to repair a chromosome scattered into fragments.

    But if there are multiple copies of the cracked chromosomes, fragments can fortuitously line up. Rather like how a jigsaw puzzle is easier to put together if there are numerous duplicates of each necessary piece, the chromosome shards can sync up and restore a functional chromosome.

    hv
    H. Volcanii grown in culture. Credit: Yejineun/Wikipedia

    “In polyploid species, the fragments generated from different copies of the chromosome overlap, and it is possible to regenerate an intact chromosome from overlapping fragments,” said Soppa.

    Desperate times, desperate measures

    To investigate if H. volcanii‘s extra chromosomes might help the archaeon survive low phosphate conditions, Soppa and colleagues starved the organism in the lab of the critical substance. The microbe continued to reproduce by splitting one cell apart into two. Interestingly, chromosome counts diminished in the “parent” and the “daughter” cells.

    “From quantifying the number of chromosomes prior to and after growth in the absence of phosphate, we have found that about 30 percent of the chromosomes are ‘missing’ afterwards,” said Soppa.

    The numbers for another potential in-house source of phosphate, H. volcanii‘s ribosomes, however, remained steady. The most likely explanation, then, of the microorganism’s hardiness when facing a phosphate nutrient shortage: H. volcanii simply cannibalizes some of its own chromosomes.

    As further verification, Soppa and colleagues tested the survival skills of H. volcanii cells that contained varying numbers of chromosome copies. Those archaea with just two copies of their chromosome turned out to be more than five times as sensitive to desiccation as those H. volcanii with a hefty complement of 20 chromosomes.

    Life, undaunted

    This newly described benefit of polyploidy in H. volcanii is a fresh demonstration of how life can make do in severe environments. So-called extremophiles have been discovered in recent decades thriving in strongly acidic hot springs, within liquid asphalt, and in other eyebrow-raising niches. Salt-tolerant bacteria and archaea, like H. volcanii, have been found to survive in deserts, simulated Mars conditions, and even the rigors of a space flight. We should not be surprised, perhaps, if life has managed to take hold on formidable worlds.

    rw
    Extremophile microbes have been found that can survive in the polluted Rio Tinto River in Spain. Mining in the river’s vicinity has led to its waters having a high heavy metal content and very low pH, though the bacteria themselves, through their metabolism, also likely contribute to the intense acidity. Image credit: Leslie Mullen

    “The understanding of how harsh the conditions can be that can be survived by some archaea and bacteria helps us to be more optimistic that life could have evolved at very rough and unsuitable places on early Earth or on other planets,” said Soppa.

    The new role ascribed to DNA, as phosphate storage, might help to explain how a completely RNA-dominated primordial era began sharing genetic duties with DNA. Life did not leap from RNA to DNA. Rather, DNA, slowly but surely, learned new tricks.

    “The hypothesis that DNA might have evolved as a storage polymer and became genetic material later, makes the step from RNA to DNA as genetic material easier, because it then would be a two-step and not a one-step process,” said Soppa. “DNA would have been around, and during long time spans additional roles could have been evolved.”

    See the full article here.
    Astrobiology Magazine is a NASA-sponsored online popular science magazine. Our stories profile the latest and most exciting news across the wide and interdisciplinary field of astrobiology — the study of life in the universe. In addition to original content, Astrobiology Magazine also runs content from non-NASA sources in order to provide our readers with a broad knowledge of developments in astrobiology, and from institutions both nationally and internationally. Publication of press-releases or other out-sourced content does not signify endorsement or affiliation of any kind.
    Established in the year 2000, Astrobiology Magazine now has a vast archive of stories covering a broad array of topics.

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  • richardmitnick 6:07 pm on June 23, 2014 Permalink | Reply
    Tags: , , , DNA, ,   

    From SLAC Lab: “Scientists Use X-rays to Look at How DNA Protects Itself from UV Light” 


    SLAC Lab

    June 23, 2014
    Andrew Gordon, agordon@slac.stanford.edu, (650) 926-2282

    The molecular building blocks that make up DNA absorb ultraviolet light so strongly that sunlight should deactivate them – yet it does not. Now scientists have made detailed observations of a “relaxation response” that protects these molecules, and the genetic information they encode, from UV damage.

    The experiment at the Department of Energy’s SLAC National Accelerator Laboratory focused on thymine, one of four DNA building blocks. Researchers hit thymine with a short pulse of ultraviolet light and used a powerful X-ray laser to watch the molecule’s response: A single chemical bond stretched and snapped back into place within 200 quadrillionths of a second, setting off a wave of vibrations that harmlessly dissipated the destructive UV energy.

    The international research team reported the results June 23 in Nature Communications.

    While protecting the genetic information encoded in DNA is vitally important, the significance of this result goes far beyond DNA chemistry, said Philip Bucksbaum, director of the Stanford PULSE Institute and a co-author of the report.

    “The new tool the team developed for this study provides a new window on the motion of electrons that control all of chemistry,” he said. “We think this will enhance the value and impact of X-ray free-electron lasers for important problems in biology, chemistry and physics.”

    Light Becomes Heat

    Researchers had noticed years ago that thymine seemed resistant to damage from UV rays in sunlight, which cause sunburn and skin cancer. Theorists proposed that thymine got rid of the UV energy by quickly shifting shape. But they differed on the details, and previous experiments could not resolve what was happening.

    The SLAC experiment took place at the Linac Coherent Light Source (LCLS), a DOE Office of Science user facility, whose bright, ultrashort X-ray laser pulses can see changes taking place at the level of individual atoms in quadrillionths of a second.

    Scientists turned thymine into a gas and hit it with two pulses of light in rapid succession: first UV, to trigger the protective relaxation response, and then X-rays, to detect and measure the response.

    “As soon as the thymine swallows the light, the energy is funneled as quickly as possible into heat, rather than into making or breaking chemical bonds,” said Markus Guehr, a DOE Early Career Program recipient and senior staff scientist at PULSE who led the study. “It’s like a system of balls connected by springs; when you elongate that one bond between two atoms and let it loose, the whole molecule starts to tremble.”

    Ejected Electrons Signal Changes

    The X-rays measured the relaxation response indirectly by stripping away some of the innermost electrons from atoms in the thymine molecule. This sets off a process known as Auger decay that ultimately ejects other electrons. The ejected electrons fly into a detector, carrying information about the nature and state of their home atoms.

    By comparing the speeds of the ejected electrons before and after thymine was hit with UV, the researchers were able to pinpoint rapid changes in a single carbon-oxygen bond: It stretched when hit with UV light and shortened 200 quadrillionths of a second later, setting off vibrations that continued for billionths of a second.

    “This is the first time we’ve been able to distinguish between two fundamental responses in the molecule – movements of the atomic nuclei and changes in the distribution of electrons – and time them within a few quadrillionths of a second,” said the paper’s first author, Brian McFarland, a postdoctoral researcher who has since moved from SLAC to Los Alamos National Laboratory.

    Guehr said the team plans more experiments to further explore the protective relaxation response and extend the new method, called time-resolved Auger spectroscopy, into other scientific realms.

    In addition to the Stanford PULSE Institute, which is a joint institute of SLAC and Stanford University, the study included researchers from LCLS, Stanford, the University of Perugia in Italy, Lawrence Berkeley National Laboratory, the University of Connecticut, Western Michigan University, the University of Gothenburg in Sweden, and UNIST in South Korea. Parts of the research were carried out at Berkeley Lab’s Advanced Light Source, a DOE Office of Science user facility. The work was funded by the DOE Office of Science, the Swedish Research Council, the Göran Gustafsson Foundation and the Knut and Alice Wallenberg Foundation.

    See the full article here.

    SLAC Campus
    SLAC is a multi-program laboratory exploring frontier questions in photon science, astrophysics, particle physics and accelerator research. Located in Menlo Park, California, SLAC is operated by Stanford University for the DOE’s Office of Science.
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  • richardmitnick 9:02 am on April 28, 2014 Permalink | Reply
    Tags: , , DNA   

    From Brookhaven Lab: “Label-free, Sequence-specific, Inexpensive Fluorescent DNA Sensors” 

    Brookhaven Lab

    April 28, 2014
    Karen McNulty Walsh

    Intensity of glow indicates level of genetic match; potentially useful for identifying microbes, harmful agents, and more

    Using principles of energy transfer more commonly applied to designing solar cells, scientists at the U.S. Department of Energy’s Brookhaven National Laboratory have developed a new highly sensitive way to detect specific sequences of DNA, the genetic material unique to every living thing. As described in a paper published in the journal Chemistry of Materials, the method is considerably less costly than other DNA assays and has widespread potential for applications in forensics, medical diagnostics, and the detection of bioterror agents.

    map
    Light-up DNA sensors: When DNA sequences are complementary, one fluorescent dye molecule (red rectangle) binds between each matching base pair. The conjugated polymer (blue) wraps around the DNA, absorbs light, and transfers the energy (via fluorescence resonant energy transfer, or FRET) to the dye to amplify the photo luminescent (PL) signal (red arrow). When DNA strands don’t match perfectly, fewer dye molecules bind and the magnitude of the PL signal is reduced.

    “This system is sensitive enough to detect individual mismatches between the bases that make up the ‘rungs’ of the twisted-ladder DNA double helix molecule, making it highly specific with no false positives.”
    — Mircea Cotlet

    “The sensors we’ve developed use a light-absorbing polymer to amplify the fluorescent signal of a dye that emits light only when it binds between two matched pieces of DNA,” said Mircea Cotlet, a physical chemist at Brookhaven’s Center for Functional Nanomaterials, who led the research and who is also an adjunct professor at Stony Brook University. The system is sensitive enough to detect individual mismatches between the bases that make up the “rungs” of the twisted-ladder DNA double helix molecule, making it highly specific with no false positives, Cotlet said.

    Plus, the method is rapid and requires no expensive equipment, just a conventional laboratory fluorimeter. It has high potential to be made field deployable for rapid analysis of crime-scene evidence and to mount a more knowledgeable, speedy response to bioterror threats.
    DNA, show thyself!

    The idea of using glowing dyes to sniff out DNA sequences is not entirely new. But finding an inexpensive fluorescent dye that inserts itself between every complementary base pair of a DNA molecule—and using a light absorbing/emitting polymer to amplify the fluorescent signal without the need for additional chemical tagging—makes the Brookhaven approach a big advance.

    two
    Zhongwei Liu, a graduate student from Stony Brook University, working with Brookhaven’s Mircea Cotlet on a new type of DNA sensor at the Center for Functional Nanomaterials.

    “The dye we use is hundreds of times cheaper than popular commercial intercalating dyes,” said Zhongwei Liu, a graduate student from Stony Brook University working with Cotlet and first author of the paper. Unbound, the green colored molecule absorbs red light but does not emit light. “But when it inserts itself in the grooves of the DNA, the dye becomes fluorescent. And so far it is the only dye that can intercalate so densely with DNA—meaning exactly one dye molecule binds between each complementary base pair of the DNA double helix”—the T-A and G-C matches that make up the genetic code.

    That means the strength of the fluorescent signal is directly related to how many dye molecules are bound—and how closely an unknown DNA sample matches a probe strand used for testing. As soon as there’s a mismatch, even at just one “rung” on the DNA ladder, a dye molecule won’t bind and the signal will weaken. Two mismatches results in a proportional drop in signal strength, and so on. “That gives us a large range for the detection of sequence mismatch,” Cotlet said.

    To amplify these signals, the scientists add a conjugated polymer. These light-absorbing materials are used for harvesting sunlight in solar cells, “but we can also make them water soluble and compatible with biomolecules like DNA,” Cotlet said.

    Synthesized by Hsing-Lin Wang, a collaborator at DOE’s Los Alamos National Laboratory, the polymers used in this study were functionalized with side chains that carry a positive charge, allowing them to naturally bind with negatively charged DNA via electrostatic interactions. “The polymer wraps and follows the helix of the DNA,” Cotlet said. “This configuration brings the polymer in close proximity with the DNA-bound dye molecules and also enhances the polymer’s ability to absorb and emit light. Both of these factors help with the transfer of energy to the dye-intercalated DNA and increase the sensitivity of the biosensor,” Cotlet said.

    So if scientists want to know whether two pieces of DNA are identical—say a known sequence from an anthrax spore and one from a suspicious-looking white powder—all they have to do is mix the samples, dye, and polymer in a test tube, turn on the light, and let the results shine for themselves. Of course, in this case, they’d be hoping to not see the light!

    This research was supported by the DOE Office of Science.

    See the full article here.

    One of ten national laboratories overseen and primarily funded by the Office of Science of the U.S. Department of Energy (DOE), Brookhaven National Laboratory conducts research in the physical, biomedical, and environmental sciences, as well as in energy technologies and national security. Brookhaven Lab also builds and operates major scientific facilities available to university, industry and government researchers. The Laboratory’s almost 3,000 scientists, engineers, and support staff are joined each year by more than 5,000 visiting researchers from around the world.Brookhaven is operated and managed for DOE’s Office of Science by Brookhaven Science Associates, a limited-liability company founded by Stony Brook University, the largest academic user of Laboratory facilities, and Battelle, a nonprofit, applied science and technology organization.
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  • richardmitnick 4:44 pm on January 29, 2014 Permalink | Reply
    Tags: , DNA, ,   

    From Berkeley Lab: “Puzzling Question in Bacterial Immune System Answered” 


    Berkeley Lab

    January 29, 2014
    Lynn Yarris (510) 486-5375 lcyarris@lbl.gov

    Berkeley Researchers Uncover the Key to Self-Awareness in Genome Editor

    A central question has been answered regarding a protein that plays an essential role in the bacterial immune system and is fast becoming a valuable tool for genetic engineering. A team of researchers with the Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of California (UC) Berkeley have determined how the bacterial enzyme known as Cas9, guided by RNA, is able to identify and degrade foreign DNA during viral infections, as well as induce site-specific genetic changes in animal and plant cells. Through a combination of single-molecule imaging and bulk biochemical experiments, the research team has shown that the genome-editing ability of Cas9 is made possible by the presence of short DNA sequences known as “PAM,” for protospacer adjacent motif.

    dna
    Short DNA sequences known as “PAM” (shown in yellow) enable the bacterial enzyme Cas9 to identify and degrade foreign DNA, as well as induce site-specific genetic changes in animal and plant cells. The presence of PAM is also required to activate the Cas9 enzyme. (Illustration by KC Roeyer.)

    “Our results reveal two major functions of the PAM that explain why it is so critical to the ability of Cas9 to target and cleave DNA sequences matching the guide RNA,” says Jennifer Doudna, the biochemist who led this study. “The presence of the PAM adjacent to target sites in foreign DNA and its absence from those targets in the host genome enables Cas9 to precisely discriminate between non-self DNA that must be degraded and self DNA that may be almost identical. The presence of the PAM is also required to activate the Cas9 enzyme.”

    With genetically engineered microorganisms, such as bacteria and fungi, playing an increasing role in the green chemistry production of valuable chemical products including therapeutic drugs, advanced biofuels and biodegradable plastics from renewables, Cas9 is emerging as an important genome-editing tool for practitioners of synthetic biology.

    “Understanding how Cas9 is able to locate specific 20-base-pair target sequences within genomes that are millions to billions of base pairs long may enable improvements to gene targeting and genome editing efforts in bacteria and other types of cells,” says Doudna who holds joint appointments with Berkeley Lab’s Physical Biosciences Division and UC Berkeley’s Department of Molecular and Cell Biology and Department of Chemistry, and is also an investigator with the Howard Hughes Medical Institute (HHMI).

    two
    Jennifer Doudna and Samuel Sternberg used a combination of single-molecule imaging and bulk biochemical experiments to show how the RNA-guided Cas9 enzyme is able to locate specific 20-base-pair target sequences within genomes that are millions to billions of base pairs long. (Photo by Roy Kaltschmdit)

    Doudna is one of two corresponding authors of a paper describing this research in the journal Nature. The paper is titled DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. The other corresponding author is Eric Greene of Columbia University. Co-authoring this paper were Samuel Sternberg, Sy Redding and Martin Jinek.

    See the full article here.

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

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