Structural biology scores with protein snapshot.

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Surface-filled representation of diacylglycerol kinase. The "porch-like" structure of the enzyme is highlighted, and the substrate diacylglycerol is depicted bound to the active site. Investigators at the Vanderbilt Center for Structural Biology used NMR methods to determine the structure of diacylglycerol kinase, the largest membrane-spanning protein studied by NMR to date. Credit: Charles Sanders, Ph.D., Vanderbilt University Center for Structural Biology.

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In a landmark technical achievement, investigators in the Vanderbilt Center for Structural Biology have used nuclear magnetic resonance (NMR) methods to determine the structure of the largest membrane-spanning protein to date.

Although NMR methods are routinely used to "take molecular pictures" of small proteins, large proteins - and particularly those that reside within the cell membrane - have been reluctant to smile for the camera.
In the June 26 issue of Science, Charles Sanders, Ph.D., professor of Biochemistry, and colleagues report the NMR structure of the large bacterial protein diacylglycerol kinase (DAGK), a complex of three subunits that each cross the membrane three times (for a total of nine membrane spans).
The group's ability to determine the NMR structure of DAGK suggests that similar methods can now be used to study the structures of other membrane proteins.
"We're taking the methods that we used for diacylglycerol kinase and applying them to high value targets such as G protein-coupled receptors," Sanders said.
G protein-coupled receptors - the largest family of cell signaling proteins - are targets for about half of all pharmaceuticals. Sanders is collaborating with other Vanderbilt investigators to tackle G protein-coupled receptor structure using both NMR and a complementary structural approach, X-ray crystallography.
DAGK may be a therapeutic target for certain types of bacterial infections. It is a virulence factor in the bacteria Streptococcus mutans, which causes tooth decay.
Sanders selected DAGK as a model for studying membrane enzymes when he started his own research lab 17 years ago. DAGK is the smallest known kinase (a protein that adds chemical groups called phosphates onto other molecules), and it is not similar to any other known proteins.
The DAGK structure, Sanders said, "confirmed that this is a really strange kinase." The enzyme has a porch-like structure, with a wide opening for its substrate diacylglycerol and the active site at the top of the porch.
"The active site looks nothing like any other kinase active site - it's a unique architecture," Sanders said.

The researchers also performed exhaustive mutagenesis studies in which they characterized mutations at each amino acid in DAGK and used the data to map the active site of the enzyme onto the structure. They identified two sets of mutations that resulted in non-functional DAGK. One set altered the active site so that it no longer did its job, and the second set caused the protein to fold incorrectly (misfolding).
Sanders said the team was surprised to find that nearly all of the mutations that caused misfolding were in the active site. The expectation, he explained, is that mutations in the active site would cause a loss of function but would not usually affect protein folding, whereas key residues for folding would be located elsewhere in the protein to underpin the scaffold for the active site.
"Our study shows that you can't make that assumption," he said.
Sanders cautions that investigators cannot simply predict the impact of a mutation based on it being located in the active site. The finding has implications for personalized medicine, which aims to use the predicted impact of disease-causing mutations to make therapy decisions.
"The therapeutic strategy for addressing catastrophic misfolding versus simple loss of function may be very different," Sanders said.
Sanders and his team, who got interested in protein folding because of their work with DAGK, are now pursuing structural studies of misfolded membrane proteins that cause diseases including peripheral neuropathy (Charcot-Marie-Tooth Disease), diabetes insipidus and Alzheimer's disease.
"For proteins that misfold because of mutations, we're using NMR tools to understand exactly what the mutations do to the proteins in terms of structure and stability," Sanders said. "We believe that understanding will lead to predictions about how to intervene and avoid misfolding."
Source: Vanderbilt University Medical Center (news : web)

TRAPping Proteins That Work Together Inside Living Cells

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ScienceDaily (June 18, 2009) — DNA might be the blueprint for living things, but proteins are the builders. Researchers trying to understand how and which proteins work together have developed a new crosslinking tool that is small and unobtrusive enough to use in live cells. Using the new tool, the scientists have discovered new details about a well-studied complex of proteins known as RNA polymerase. The results suggest the method might uncover collaborations between proteins that are too brief for other techniques to pinpoint.

"Conventional methods used to find interacting proteins have limitations that we are trying to circumvent," said biochemist Uljana Mayer of the Department of Energy's Pacific Northwest National Laboratory. "They also create conditions that are different from those inside cells, so you can't find all the interactions that proteins would normally engage in."
Proteins are the workhorses in an organism's cells. Whole fields of research are dedicated to teasing out which proteins work together to make cells function. For example, drug researchers seek chemicals that disrupt or otherwise change how proteins interact to combat diseases; environmental scientists need to understand how proteins collaborate in ecosystems to make them thrive or fail.
To learn about protein networks, scientists start with a familiar one and use it as bait to find others that work alongside it. To pin down the collaborators, researchers make physical connections between old and new proteins with chemicals called crosslinkers. The sticky crosslinkers will only connect proteins close enough to work together, the thinking goes. But most crosslinkers are too large to squeeze into living cells, are harmful to cells, or link proteins that are neighbors but not coworkers.
To address these issues, Mayer and her PNNL colleagues developed a crosslinking method that uses small crosslinkers whose stickiness can be carefully controlled. To find coworkers of a protein of interest, Mayer and her colleagues build a tiny molecule called a tag into the initial protein. They then add a small molecule called TRAP to the living cell, which finds and fits into the tag like two pieces in a puzzle. TRAP waves around, bumping into nearby proteins. The scientists control TRAP with a flash of light, causing it to stick to coworkers it bumps into. The researchers then identify the new "TRAPped" proteins in subsequent analyses.
To demonstrate how well this method works, Mayer and colleagues tested it out on RNA polymerase, a well-studied machine in cells. The polymerase is made up of many proteins that cooperate to translate DNA. One of the polymerase proteins has a tail that is known to touch the DNA and some helper proteins just before the polymerase starts translating. No one knew if this tail -- also known as the C-terminus of the alpha subunit -- touches anything else in the core of the RNA polymerase complex.
The team engineered a tag in the C-terminus and cultured bacteria with the tagged RNA polymerase. After adding TRAP to the cells and giving it time to find the C-terminus tag, the team shined a light on the cultures.
The team then identified the proteins marked with TRAP using instruments in EMSL, DOE's Environmental Molecular Sciences Laboratory on the PNNL campus. They found that the tagged protein, as expected, interacts with many other proteins, for example previously identified helper proteins, so-called transcription factors. But they also found it on another core protein called the beta subunit, suggesting the tail of the alpha subunit makes contact with the beta subunit as it plugs along. This interaction had never been seen before.
"No one knows what the polymerase looks like when it is running," said Uljana Mayer. "Here we see the C-terminus swings back to grab the beta subunit once the polymerase starts working."
The team report their results June 15 in the journal ChemBioChem. The tag in their unique method is made up of a "tetracysteine motif" -- two pairs of the amino acid cysteine separated by two other amino acids that doesn't interfere with the normal function of the protein of interest. TRAP includes a small "biarsenical" probe, which fluoresces so the team can find the proteins to which it has become attached. TRAP can also be easily unlinked from the tag with a simple biochemical treatment, allowing researchers to piece out the coworker from their original protein of interest.
The team also tested the method on other proteins, such as those found in young muscle cells. Mayer said they will use the method in the future to understand how environmental conditions affect how proteins work together in large networks.
Journal reference:
P. Yan, T. Wang, G.J. Newton, T.V. Knyushko, Y. Xiong, D. J. Bigelow, T.C. Squier, and M.U. Mayer. A Targeted Releasable Affinity Probe (TRAP) for In Vivo Photocrosslinking. ChemBioChem, 2009; 10: 1507-1518 DOI: 10.1002/cbic.200900029
Adapted from materials provided by DOE/Pacific Northwest National Laboratory.

Nanocrystals Reveal Activity Within Cells

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ScienceDaily (June 18, 2009) — Researchers at the U.S. Department of Energy’s (DOE) Lawrence Berkeley National Laboratory have created bright, stable and bio-friendly nanocrystals that act as individual investigators of activity within a cell. These ideal light emitting probes represent a significant step in scrutinizing the behaviors of proteins and other components in complex systems such as a living cell.

Labeling a given cellular component and tracking it through a typical biological environment is fraught with issues: the probe can randomly turn on and off, competes with light emitting from the cell, and often requires such intense laser excitation, it eventually destroys the probe, muddling anything you’d be interested in seeing.
“The nanoparticles we’ve designed can be used to study biomolecules one at a time,” said Bruce Cohen, a staff scientist in the Biological Nanostructures Facility at Berkeley Lab’s nanoscience research center, the Molecular Foundry. “These single-molecule probes will allow us to track proteins in a cell or around its surface, and to look for changes in activity when we add drugs or other bioactive compounds.”
Molecular Foundry post-doctoral researchers Shiwei Wu and Gang Han, led by Cohen, Imaging and Manipulation of Nanostructures staff scientist Jim Schuck and Inorganic Nanostructures Facility Director Delia Milliron, worked to develop nanocrystals containing rare earth elements that absorb low-energy infrared light and transform it into visible light through a series of energy transfers when they are struck by a continuous wave, near-infrared laser. Biological tissues are more transparent to near-infrared light, making these nanocrystals well suited for imaging living systems with minimal damage or light scatter.
“Rare earths have been known to show phosphorescent behavior, like how the old-style television screen glows green after you shut it off. These nanocrystals draw on this property, and are a million times more efficient than traditional dyes,” said Schuck. “No probe with ideal single-molecule imaging properties had been identified to date—our results show a single nanocrystal is stable and bright enough that you can go out to lunch, come back, and the intensity remains constant.”
To study how these probes might behave in a real biological system, the Molecular Foundry team incubated the nanocrystals with embryonic mouse fibroblasts, cells crucial to the development of connective tissue, allowing the nanocrystals to be taken up into the interior of the cell. Live-cell imaging using the same near-infrared laser showed similarly strong luminescence from the nanocrystals within the mouse cell, without any measurable background signal.
“While these types of particles have existed in one form or another for some time, our discovery of the unprecedented ’single-molecule’ properties these individual nanocrystals possess opens a wide range of applications that were previously inaccessible,” Schuck adds.
“Non-blinking and photostable upconverted luminescence from single lanthanide-doped nanocrystals,” by Shiwei Wu, Gang Han, Delia J. Milliron, Shaul Aloni, Virginia Altoe, Dmitri Talapin, Bruce E. Cohen and P. James Schuck, appears in Proceedings of the National Academy of Sciences and is available in Proceedings of the National Academy of Sciences online.
Work at the Molecular Foundry was supported by the Office of Basic Energy Sciences within the DOE Office of Science.
Adapted from materials provided by DOE/Lawrence Berkeley National Laboratory.

Scientists Show Bacteria Can 'Learn' And Plan Ahead

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ScienceDaily (June 18, 2009) — Bacteria can anticipate a future event and prepare for it, according to new research at the Weizmann Institute of Science. In a paper that appeared June 17 in Nature, Prof. Yitzhak Pilpel, doctoral student Amir Mitchell and research associate Dr. Orna Dahan of the Institute's Molecular Genetics Department, together with Prof. Martin Kupiec and Gal Romano of Tel Aviv University, examined microorganisms living in environments that change in predictable ways.

Their findings show that these microorganisms' genetic networks are hard-wired to 'foresee' what comes next in the sequence of events and begin responding to the new state of affairs before its onset.
E. coli bacteria, for instance, which normally cruise harmlessly down the digestive tract, encounter a number of different environments on their way. In particular, they find that one type of sugar – lactose – is invariably followed by a second sugar – maltose – soon afterward. Pilpel and his team of the Molecular Genetics Department, checked the bacterium's genetic response to lactose, and found that, in addition to the genes that enable it to digest lactose, the gene network for utilizing maltose was partially activated. When they switched the order of the sugars, giving the bacteria maltose first, there was no corresponding activation of lactose genes, implying that bacteria have naturally 'learned' to get ready for a serving of maltose after a lactose appetizer.
Another microorganism that experiences consistent changes is wine yeast. As fermentation progresses, sugar and acidity levels change, alcohol levels rise, and the yeast's environment heats up. Although the system was somewhat more complicated that that of E. coli, the scientists found that when the wine yeast feel the heat, they begin activating genes for dealing with the stresses of the next stage. Further analysis showed that this anticipation and early response is an evolutionary adaptation that increases the organism's chances of survival.
Ivan Pavlov first demonstrated this type of adaptive anticipation, known as a conditioned response, in dogs in the 1890s. He trained the dogs to salivate in response to a stimulus by repeatedly ringing a bell before giving them food. In the microorganisms, says Pilpel, 'evolution over many generations replaces conditioned learning, but the end result is similar.' 'In both evolution and learning,' says Mitchell, 'the organism adapts its responses to environmental cues, improving its ability to survive.' Romano: 'This is not a generalized stress response, but one that is precisely geared to an anticipated event.'
To see whether the microorganisms were truly exhibiting a conditioned response, Pilpel and Mitchell devised a further test for the E. coli based on another of Pavlov's experiments. When Pavlov stopped giving the dogs food after ringing the bell, the conditioned response faded until they eventually ceased salivating at its sound. The scientists did something similar, using bacteria grown by Dr. Erez Dekel, in the lab of Prof. Uri Alon of the Molecular Cell Biology Department, in an environment containing the first sugar, lactose, but not following it up with maltose. After several months, the bacteria had evolved to stop activating their maltose genes at the taste of lactose, only turning them on when maltose was actually available.
'This showed us that there is a cost to advanced preparation, but that the benefits to the organism outweigh the costs in the right circumstances,' says Pilpel. What are those circumstances? Based on the experimental evidence, the research team created a sort of cost/benefit model to predict the types of situations in which an organism could increase its chances of survival by evolving to anticipate future events. They are already planning a number of new tests for their model, as well as different avenues of experimentation based on the insights they have gained.
Pilpel and his team believe that genetic conditioned response may be a widespread means of evolutionary adaptation that enhances survival in many organisms – one that may also take place in the cells of higher organisms, including humans. These findings could have practical implications, as well. Genetically engineered microorganisms for fermenting plant materials to produce biofuels, for example, might work more efficiently if they gained the genetic ability to prepare themselves for the next step in the process.
Prof. Yitzhak Pilpel's research is supported by the Ben May Charitable Trust and Madame Huguette Nazez, Paris, France.
Adapted from materials provided by Weizmann Institute of Science, via EurekAlert!, a service of AAAS.

Long-standing Mystery Of How Plants Make Eggs Solved

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ScienceDaily (June 4, 2009) — A long-standing mystery surrounding a fundamental process in plant biology has been solved by a team of scientists at the University of California, Davis.

The group’s groundbreaking discovery that a plant hormone called auxin is responsible for egg production has several major implications.
First, this is the first definitive report of a plant hormone acting as a morphogen, that is, a substance that directs the pattern of development of cells based on its concentration.
Also, the study’s results provide tantalizing new insights into the evolutionary pathway that flowering plants took 135 million years ago when they split off from gymnosperms, the “naked-seeded” plant group that includes conifers, cycads and ginkgo trees.
Finally, the group used their discovery to make additional egg cells within plant reproductive structures, raising the prospects that these techniques may someday be used for enhancing the reproduction and fertility of crop plants.
“So the sequence becomes clear now,” said Venkatesan Sundaresan, the UC Davis professor of plant biology and plant sciences who led the study. “The plant triggers auxin synthesis at one end of the female reproductive unit called the embryo sac, creating an auxin gradient. The eight nuclei in the sac are then exposed to different levels of auxin, but only the nucleus in the correct position in the gradient becomes an egg cell. And that cell is subsequently fertilized to make the next generation.”
A paper describing the study was published June 4 in the journal Science’s online site, Science Express, in advance of its publication in the journal later this month.
Development of sperm and egg cells in plants
In humans and other animals, the germ cells for production of eggs and sperm are established at birth. But cells in flowering plants are assigned more or less randomly to become reproductive units when the plant reaches sexual maturity. Within the flower, sperm cells are produced by pollen at the tips of stamens, while egg cells develop in ovules, tiny structures embedded in the ovary at the base of the pistil.
At the start of the process of egg-cell development, a “mother cell” in the ovule divides several times, in a sequence involving both meiosis and mitotic divisions. These divisions result in the creation of an oblong, cell-like structure called the embryo sac, which contains eight nuclei, three of which are clustered near the open end of the ovule.
Within hours cell membranes start forming, eventually, creating seven cells: the all-important egg cell near the ovule opening where pollen will enter, and six other supporting cells, with essential functions for seed formation.
“The big question in our field for the past 50 years or more has been: How does this process happen in such a beautifully orchestrated pattern?” Sundaresan said. “It’s been clear that there’s a program here telling the plants exactly what to do, and that it is working not on cells, but on nuclei.”
Auxin concentrations determine fate of nuclei
Two years ago Sundaresan and a postdoctoral fellow in his laboratory, Gabriela Pagnussat, used genetic tools to shift the position of a single nucleus at one end of an embryo sac in the plant Arabidopsis. When they examined the mature sac, they found that it had produced two egg cells instead of one.
Sundaresan recognized that a pattern shift like this was similar to the response that had been reported two decades earlier in Drosophila fruit flies in experiments that provided the first direct evidence for the existence of morphogens.
This prompted him to begin searching for a substance in Arabadopsis that might be acting as a morphogen. When the group discovered that auxin was accumulating at the open end of the ovule, they turned their attention to this ubiquitous hormone, which is known to play myriad signaling roles in plant growth and behavioral processes. (The hormone’s existence was first guessed by Charles Darwin when he was studying how plants grow towards light.)
After many tests, Sundaresan and his group found that during embryo sac formation, auxin concentrations did indeed follow a gradient, with the highest levels occurring in the ovule at the end of the embryo sac where the pollen enters and lowest levels occurring at the opposite end of the sac.
To test the theory that this gradient was determining the fate of nuclei in the sac, Sundaresan and his group created a series of genetically manipulated Arabadopsis plants. In some plants they ratcheted up production of auxin in the embryo sac, and in others they decreased the sac’s sensitivity to auxin, creating the same effect that a decline in auxin would make.
When they examined these experimental plants, their hypothesis was confirmed: Auxin concentrations determined the fate of the nuclei. Knowing whether auxin levels were high or low, it became possible to predict the appearance or disappearance of egg cells at different positions within the embryo sac.
Finally, the group employed a long series of bio-manipulative techniques to determine that the auxin gradient they had discovered within the embryo sac was due to on-site synthesis rather than transport from a source outside the sac.
“What we have found about the way auxin works here is amazing,” Sundaresan said. “The idea that you can have a small molecule like this being maintained in a gradient within this eight-nucleate structure through synthesis alone is mind-boggling.”
Implications for flowering plant evolution
Development of the embryo sac is arguably the key element in the evolution from gymnosperms to flowering plants, also known as angiosperms.
Yet the fossil record reveals very little about the stages that led from gymnosperm seed production to angiosperm seed production when the transition occurred around 135 million years ago. The rapid expansion of flowering plants and their eventual domination of the Earth’s vegetation was called “an abominable mystery” by Darwin.
By elucidating the mechanism of embryo sac development, Sundaresan and his team have opened the door to new work into the evolutionary pathway between these two major plant groups. The discovery supports what is known as the modular theory, which posits that the first angiosperms underwent a drastic reduction of their female reproductive unit compared to the gymnosperms, allowing flowering plants to reproduce more efficiently and eventually supplant their naked-seeded forebears.
Most remarkably, perhaps, the new work suggests that the eight nuclei of the angiosperm embryo sac have retained developmental plasticity in their evolution from gymnosperms. “It’s amazing that even though the split supposedly happened over a hundred million years ago,” Sundaresan said, “all these nuclei still have the capacity to become egg cells.”
Collaborators in the study are lead author Gabriela Pagnussat and Monica Alandete-Saez, who were postdoctoral researchers with Sundaresan when they did the work, and John L. Bowman, a professor of plant biology at UC Davis at the time of the study, now at Monash University in Melbourne, Australia.
The work was supported by grants from the National Science Foundation.
Adapted from materials provided by University of California - Davis.

Geography And History Shape Genetic Differences In Humans

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ScienceDaily (June 5, 2009) — New research indicates that natural selection may shape the human genome much more slowly than previously thought. Other factors -- the movements of humans within and among continents, the expansions and contractions of populations, and the vagaries of genetic chance – have heavily influenced the distribution of genetic variations in populations around the world.

The study, conducted by a team from the Howard Hughes Medical Institute, the University of Chicago, the University of California and Stanford University, is published June 5 in the open-access journal PLoS Genetics.
In recent years, geneticists have identified a handful of genes that have helped human populations adapt to new environments within just a few thousand years—a strikingly short timescale in evolutionary terms. However, the team found that for most genes, it can take at least 50,000-100,000 years for natural selection to spread favorable traits through a human population. According to their analysis, gene variants tend to be distributed throughout the world in patterns that reflect ancient population movements and other aspects of population history.
"We don't think that selection has been strong enough to completely fine-tune the adaptation of individual human populations to their local environments," says co-author Jonathan Pritchard. "In addition to selection, demographic history -- how populations have moved around -- has exerted a strong effect on the distribution of variants."
To determine whether the frequency of a particular variant resulted from natural selection, Pritchard and his colleagues compared the distribution of variants in parts of the genome that affect the structure and regulation of proteins to the distribution of variants in parts of the genome that do not affect proteins. Since these neutral parts of the genome are less likely to be affected by natural selection, they reasoned that studying variants in these regions should reflect the demographic history of populations.
The researchers found that many previously identified genetic signals of selection may have been created by historical and demographic factors rather than by selection. When the team compared closely related populations they found few large genetic differences. If the individual populations' environments were exerting strong selective pressure, such differences should have been apparent.
Selection may still be occurring in many regions of the genome, says Pritchard. But if so, it is exerting a moderate effect on many genes that together influence a biological characteristic. "We don't know enough yet about the genetics of most human traits to be able to pick out all of the relevant variation," says Pritchard. "As functional studies go forward, people will start figuring out the phenotypes that are associated with selective signals," says lead author Graham Coop. "That will be very important, because then we can figure out what selection pressures underlie these episodes of natural selection."
But even with further research, much will remain unknown about the processes that have resulted in human traits. In particular, Pritchard and Coop urge great caution in trying to link selection with complex characteristics like intelligence. "We're in the infancy of trying to understand what signals of selection are telling us," says Coop, "so it's a very long jump to attribute cultural features and group characteristics to selection."
Journal reference:
Coop G, Pickrell JK, Novembre J, Kudaravalli S, Li J, et al. The Role of Geography in Human Adaptation. PLoS Genetics, 2009; 5 (6): e1000500 DOI: 10.1371/journal.pgen.1000500
Adapted from materials provided by Public Library of Science, via EurekAlert!, a service of AAAS.

Breakthrough in the treatment of bacterial meningitis

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It can take just hours after the symptoms appear for someone to die from bacterial meningitis. Now, after years of research, experts at The University of Nottingham have finally discovered how the deadly meningococcal bacteria is able to break through the body's natural defence mechanism and attack the brain.

The discovery could lead to better treatment and vaccines for meningitis and could save the lives of hundreds of children.
Bacterial meningitis in childhood is almost exclusively caused by the respiratory tract pathogens Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae. The mechanism used by these lethal germs to break through the blood brain barrier (BBB) has, until now, been unknown.
The team led by Dlawer Ala'Aldeen, Professor of Clinical Microbiology and Head of the Molecular Bacteriology and Immunology Group at the Centre for Biomolecular Sciences, recently discovered that all three pathogens target the same receptor on human cerebrovascular endothelial cells — the specialised filtering system that protects our brain from disease — enabling the organisms to cross the blood-brain barrier.
Their findings, published today in The Journal of Clinical Investigation, suggest that disruption or modulation of this interaction of bacterial adhesins with the receptor might offer unexpectedly broad protection against bacterial meningitis and may provide a therapeutic target for the prevention and treatment of disease.
Professor Ala'Aldeen, who has been studying meningitis and its causes for over 20 years, said: "This is a significant breakthrough which will help us design novel strategies for the prevention and treatment of bacterial meningitis. Identification of the human receptor and bacterial ligands is like identifying a mysterious key and its lock, which will open new doors and pave the way for new discoveries."
The research, carried out in collaboration with the Department of Infectious Diseases at St. Jude Children's Research Hospital in Memphis Tennessee, also involved students from the University who have been regular and willing volunteers in the research programme.
Professor Ala'Aldeen said: "The ultimate aim is to save lives by protecting the healthy and curing the sick. We are one step closer to new breakthroughs that would prevent disease or its complications. There still is a long way to go before we have the ultimate vaccine and the ultimate treatment of bacterial meningitis."
Source: University of Nottingham (news : web)

Developed a human monoclonal antibody that neutralizes the Hepatitis C virus (HCV).

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ScienceDaily (May 11, 2009) — aking aim at a leading cause of liver failure in the United States, a team of scientists at the Massachusetts Biologic Laboratories (MBL) of the University of Massachusetts Medical School (UMMS) has developed a human monoclonal antibody that neutralizes the Hepatitis C virus (HCV). The new antibody effectively neutralized the virus in culture, and then prevented infection by the virus in a pre-clinical animal model of the disease.

Details of the research were presented April 23 in Copenhagen, Denmark at the 44th Annual Meeting of the European Association for the Study of the Liver (EASL). "We are pleased with the progress of this program," said Donna Ambrosino, MD, executive director of the MBL and a professor of pediatrics at the Medical School. "This antibody shows significant efficacy against the virus."
In the current study, MBL scientists injected transgenic mice (HuMAb Mouse® technology, Medarex, Inc.) with elements of HCV and then painstakingly searched for individual human antibodies produced in the mice that would recognize and bind to the HCV's outer coat, known as the glycoprotein. Once they found human antibodies that looked promising, they evaluated in vitro the ability of those antibodies to neutralize the virus and selected a lead candidate antibody for further characterization. Collaborative work with clinical researchers from the Department of Medicine at the Medical School's Worcester campus demonstrated that this antibody, now known as MBL-HCV1, was able to bind tightly with all genotypes of HCV tested from infected patient samples.
MBL-HCV1 was then tested off-site on three non-human primates. In that study, one animal received no antibody, one a low dose of the new antibody, and one a higher dose. Then all three animals were exposed to HCV. The animals with low or no antibody dosages developed HCV infections, but the animal with the higher dose was protected. Subsequently, researchers gave the high-dose of the antibody to the animal that originally received no antibody, and in that case the HCV was cleared from that animal's system. "These results are encouraging as a possible treatment for HCV infected patients, but more work needs to be done before we know how effective it will be in people," Dr. Ambrosino noted.
HCV attacks the liver and can eventually lead to liver failure. According to the U.S. Centers for Disease Control and Prevention, 3.2 million Americans are chronically infected with HCV and some 10,000 die annually of the disease. Globally, as many as 170 million people are estimated to suffer from HCV infection. For the most serious cases of HCV that do not respond to antiviral drugs, liver transplantation is the only option.
Typically 2,000 to 4,000 liver transplants are done each year in the United States (far less than the number of people on the waiting list for available organs). Transplantation can be a life saving treatment; however, in nearly all cases the patient's new liver is eventually infected by HCV because the virus remains in the patient's bloodstream during surgery. The powerful antiviral drugs now used to attack HCV prior to end-stage liver failure are not routinely used during surgery due to the patients' weakened condition and because of the strong medication used to avoid rejection of the new liver. After re-infection with HCV, nearly 40 percent of patients suffer rapid liver failure.
To close that clinical gap, the new antibody developed at MBL is designed to be a therapy shortly before and after transplant surgery. By giving a patient the new antibody before and during the time when the donor liver is implanted, researchers hope the HCV virus left in the bloodstream will be neutralized and rendered unable to infect the new liver. Then, because monoclonal antibodies are highly specific and typically have little or no side-effects, additional dosages of the new antibody could, theoretically, be given immediately after transplant surgery to continue neutralizing any remaining virus.
It is also possible, researchers theorize, that the antibody could be used in combination with new antiviral drugs for treatment in patients with newly diagnosed HCV infection. Use of the new antibody for both liver transplant patients and in newly diagnosed HCV patients will now be further evaluated. A Phase 1 human clinical trial of MBL-HCV1 in healthy subjects is expected to begin later this year.
Adapted from materials provided by University of Massachusetts Medical School, via EurekAlert!, a service of AAAS.

Biotechnology: Engineered Moss Can Produce Human Proteins

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ScienceDaily (May 11, 2009) — ETH Zurich researchers have shown that mosses and humans share unexpected common characteristics. These evolutionary relics could be useful in the production of therapeutic proteins.

At first glance, mosses and human beings have little in common. The moss Physcomitrella patens is small, pale green, immobile, and uses sunlight as its energy source. Humans are large, mobile, and need to obtain energy by eating vegetable or animal foods.
Transferring mammalian genes into moss
This made the result of the experiments carried out by researchers in the group led by Martin Fussenegger, Professor of Chemical and Bioengineering at ETH Zurich, all the more astonishing. In collaboration with researchers at the University of Freiburg im Breisgau, the PhD student Marc Gitzinger carried out tests to see what happens when unmodified human or mammalian genes are inserted into the moss genome. They transferred the foreign, unmodified genes into the moss and discovered that the moss was easily able to manufacture the proteins encoded therein.
This cannot be taken for granted, since the same process does not work when a mammalian gene is implanted into what are known as “higher” flowering plants. The reason is that sections of the start and finish sequences of the genes of animals, plants, fungi and bacteria are considerably different. They are responsible for ensuring that a gene in the organism is recognized as such, and the proteins encoded by it are produced in the correct amount and are released from the cell. The more remote the relationship between living organisms, the greater the difference between these sequences. This is why biotechnologists must normally adapt them to a foreign organism before transplanting a gene into it. The researchers were astonished to find that this was not necessary in the case of the moss.
Moss as a generalist
The explanation given for this by Ralf Reski, Professor of Plant Biotechnology at the University of Freiburg im Breisgau, is that the moss has remained a generalist. It underwent the last major modification about 450 million years ago when it changed from living in water to a life on land, adapting to the new living conditions and then remaining unaltered for millions of years, both in its appearance and at a genetic level.
The process used by the moss to produce its proteins is less sophisticated than in “higher” organisms. In contrast to the moss, these latter organisms underwent major further developments and specializations over the course of 450 million years. On the other hand, the moss clearly retained – for millions of years – the ability to read foreign genes such as those from mammals and thus also from humans, and to translate them into proteins, probably without ever having made any use of this capability during these 450 million years.
A cost-effective alternative to mammalian cells
Today, the moss Physcomitrella patens and its ability to manufacture mammalian proteins could help to satisfy the large worldwide demand for therapeutic proteins. One well-known example is insulin, which enables diabetics to control their blood sugar level.
Nowadays, therapeutic proteins are mainly manufactured in mammalian cells, which are very expensive to culture. They need to be maintained at body temperature with a continuous supply of nutrients and oxygen, and the production process is costly. At present, global production capacity cannot match the demand. Because of the difficulties involved in handling them, production is possible only in industrialized countries.
In contrast, the moss Physcomitrella patens is comparatively undemanding. It needs water, a couple of nutrient salts and some light to allow it to flourish and produce proteins. This makes it convenient and simple to handle in a bioreactor, and, in the future, it might enable even less developed nations to satisfy their requirement for therapeutic proteins. However, further research will be needed before the moss can be used to produce therapeutic proteins on an industrial scale.
Journal reference:
Gitzinger et al. Functional cross-kingdom conservation of mammalian and moss (Physcomitrella patens) transcription, translation and secretion machineries. Plant Biotechnology Journal, 2009; 7 (1): 73 DOI: 10.1111/j.1467-7652.2008.00376.x
Adapted from materials provided by ETH Zurich.

Sexually Transmitted Infections: Transistors Used To Detect Fungus Candida Albicans

SOURCE

ScienceDaily (May 11, 2009) — The Nanosensors group from the Universidad Rovira i Virgili has created a biosensor, an electrical and biological device, which is able to selectively detect the Candida albicans yeast in very small quantities of only 50 cfu/ml (colony-forming units per millilitre).

"The technique uses field-effect transistors (electronic devices that contain an electrode source and a draining electrode connected to a transducer) based on carbon nanotubes and with Candida albicans-specific antibodies", Raquel A. Villamizar, lead author of the study said.
The Candida samples, which can be obtained from blood, serum or vaginal secretions, are placed directly on the biosensor, where the interaction between antigens and antibodies changes the electric current of the devices. This change is recorded and makes it possible to measure the amount of yeast present in a sample.
"Thanks to the extraordinary charge transference properties of the carbon nanotubes, the fungus detection process is direct, fast, and does not require the use of any marker", remarks Villamizar, who is co-author of a study that provides details of the biosensor and was published recently in the journal Sensors and Actuators B: Chemical.
To date, conventional diagnosis of Candida has been carried out using microbial cultures, serological tests, PCR molecular biology techniques (polymerase chain reactions used to amplify DNA), or immunoassays such as ELISA (Enzyme Linked Inmunoabsorbent Assay).
These techniques require long analysis times and sometimes give rise to false positives and negatives. ELISA also requires the use of markers (compounds that must be added to detect the presence of yeast by fluorescence and other techniques).
The new carbon nanotubes biosensor, however, "makes it possible to improve some of the quality parameters of the traditional methods, for example the speed and simplicity of measurements, and it is an alternative tool that could be used in routine sample analysis", explains Villamizar.
The researcher adds that by using this biosensor "it will be possible in future to obtain a rapid diagnosis of infection with this pathogen, which will help to ensure administration of the correct prophylactic treatments".
The Candida albicans fungus exists naturally in the skin, mouth, the mucous membranes lining the digestive tract, and the respiratory and genitourinary systems. This yeast can cause anything from simple mycosis of the skin to complicated cases of candidiasis. It is much more commonly found in patients suffering from immunodeficiency, tumours, diabetes and lymphomas, among other diseases.
Journal reference:
Villamizar et al. Improved detection of Candida albicans with carbon nanotube field-effect transistors. Sensors and Actuators B Chemical, 2009; 136 (2): 451 DOI: 10.1016/j.snb.2008.10.013
Adapted from materials provided by Plataforma SINC.

Deep in the Red: Using Infrared to Watch What Goes On in a Living Body

SOURCE

An infrared version of the Nobel Prize-winning green fluorescent protein could make the technique even more powerful.

Fluorescent proteins, which are compounds that can absorb and then emit light, have become a powerful instrument in the cell biologist's toolkit—so powerful, in fact, that the discovery and development of green fluorescent proteins from jellyfish earned the 2008 Nobel Prize in Chemistry. (Here's a Q&A with one of the winners, Columbia University's Martin Chalfie, about his work.) These proteins have limitations, however: They need to be excited with the blue to orange part of the visible spectrum, at wavelengths of 495 to 570 nanometers. These wavelengths of light are too short to penetrate tissue very well, and so green fluorescent proteins are mainly used in test tube studies to watch cell division or to label certain cell types.
But one of the 2008 Nobelists, Roger Y. Tsien of the University of California, San Diego, and his U.C.S.D. colleagues report in today's issue of Science that they have developed a new fluorescent protein that could enable scientists to tag and visualize cellular activity as it happens inside a live animal. The protein, after absorbing light from the far-red part of the spectrum, shines in the near-infrared, at wavelengths of around 700 nanometers.
These longer wavelengths can penetrate mammalian tissue and even pass through bone. "Say you label a tumor with a green fluorescent protein, and if this labeled tumor is buried inside the animal, then you barely can get green fluorescence out," says lead researcher Xiaokun Shu. "But if you label this deeply buried tumor by infrared fluorescent proteins, you will get a stronger signal because infrared penetrates tissue more efficiently."
Tsien's group derived the infrared fluorescent protein from a hardy bacterium called Deinococcus radiodurans, famous for its ability to survive extreme environments. Bacteria do not actually use this class of proteins, called bacteriophytochromes, to emit light. "They use these bacteriophytochromes to control gene expression,” Shu says—the proteins convert absorbed light into energy to signal certain genes to turn on or off.
The initial challenge for researchers was to re-engineer the protein so that absorbed light would be re-emitted instead of being used as a source of power. They accomplished the feat by deleting the part of the protein that converts the absorbed light into chemical energy; as a result, this truncated and mutant form gave up its absorbed energy as an infrared glow. The scientists incorporated the engineered bacterial protein into mammalian cells—specifically, into the liver of a live mouse, which lit up with infrared light.
The finding paves the way for in vivo visualization of a wide range of biochemical processes and internal organs in animals. (Its use in humans is unlikely, as it would require gene therapy and the ethically dubious transplantation of bacterial genes into humans.)
“This is so important," comments David James, a cell biologist from Australia's Garvan Institute in Sydney, "because a lot of knowledge at the moment is confined to individual cells grown on a glass coverslip," leaving open the question of whether that knowledge "is transferable to an animal." The infrared version could also solve the problem of naturally occuring fluorescence from other biological molecules, which tend to glow at wavelengths similar to conventional fluorescent protein markers and thereby create a lot of “background noise," James says.

But even greater potential lies in harnessing the bacteriophytochrome's original function, namely, powering gene expression. It should be feasible, Tsien thinks, to put back in the signal-controlling properties of the phytochrome. Then it could be possible with animals to “switch on genes and control biochemistry" with light, he says.
For example, you want to explore the effects on mouse behavior of switching on a particular gene that controls some aspect of brain function, but, thankfully for the mouse, you do not want to open up its skull or stick a needle in its brain. "If the infrared fluorescent protein can be made to turn back into an infrared phytochrome, you could have the switch all ready and just waiting for enough infrared light," Tsien speculates. Because infrared light can penetrate the skull, it can reach the phytochrome and remotely switch the gene on, resulting in observable changes in the mouse's behavior.
It's the next evolutionary step for fluorescent proteins, remarks Tsien, who believes that phytochromes represent a class of proteins with enormous potential. If he's correct, then in the coming years, expect more scientists to see the (infrared) light.

By Bianca Nogrady

New Method Can Reveal Ancestry Of All Genes Across Many Different Genomes

Source:

http://www.sciencedaily.com/releases/2007/09/070911155206.htm

Science Daily — The wheels of evolution turn on genetic innovation -- new genes with new functions appear, allowing organisms to grow and adapt in new ways. But deciphering the history of how and when various genes appeared, for any organism, has been a difficult and largely intractable task.

Now a team led by scientists at the Broad Institute of MIT and Harvard has broken new ground by developing a method, described in the September 6 advance online edition of Nature, that can reveal the ancestry of all genes across many different genomes. First applied to 17 species of fungi, the approach has unearthed some surprising clues about why new genes pop up in the first place and the biological nips and tucks that bolster their survival.
"Having the ability to trace the history of genes on a genomic scale opens the doors to a vast array of interesting and largely unexplored scientific questions," said senior author Aviv Regev, an assistant professor of biology at MIT and a core member of the Broad Institute. Although the principles laid out in the study pertain to fungi, they could have relevance to a variety of other species as well.
It has been recognized for decades that new genes first arise as carbon copies of existing genes. It is thought that this replication allows one of the gene copies to persist normally, while giving the other the freedom to acquire novel biological functions. Though the importance of this so-called gene duplication process is well appreciated -- it is the grist for the mill of evolutionary change -- the actual mechanics have remained murky, in part because scientists have lacked the tools to study it systematically.
Driven by the recent explosion of whole genome sequence data, the authors of the new study were able to assemble a natural history of more than 100,000 genes belonging to a group of fungi known as the Ascomycota. From this, the researchers gained a detailed view of gene duplication across the genomes of 17 different species of fungi, including the laboratory model Saccharomyces cerevisiae, commonly known as baker's yeast.
The basis for the work comes from a new method termed "SYNERGY", which first author Ilan Wapinski and his coworkers developed to help them reconstruct the ancestry of each fungal gene. By tracing a gene's lineage through various species, the method helps determine in which species the gene first arose, and if -- and in what species -- it became duplicated or even lost altogether. SYNERGY draws its strength from the use of multiple types of data, including the evolutionary or "phylogenetic" tree that depicts how species are related to each other, and the DNA sequences and relative positions of genes along the genome.
Perhaps most importantly, the method does not tackle the problem of gene origins in one fell swoop, as has typically been done, but rather breaks it into discrete, more manageable bits. Instead of treating all species at once, SYNERGY first focuses on a pair of the most recently evolved species -- those at the outer branches of the tree -- and works, two-by-two, toward the more ancestral species that comprise the roots.
From this analysis, Regev and her colleagues were able to identify a set of core principles that govern gene duplication in fungi. The findings begin to paint a picture of how new genes are groomed over hundreds of millions of years of evolution.
The study was supported by grants from the Burroughs Wellcome Fund and the National Institute of General Medical Sciences.
Note: This story has been adapted from a news release issued by Massachusetts Institute Of Technology.

Fausto intilla

www.oloscience.com

Synthetic Biology? Memory In Yeast Cells Synthesized

Source:

http://www.sciencedaily.com/releases/2007/09/070915081448.htm

Science Daily — Harvard Medical School researchers have successfully synthesized a DNA-based memory loop in yeast cells, findings that mark a significant step forward in the emerging field of synthetic biology.
After constructing genes from random bits of DNA, researchers in the lab of Professor Pamela Silver, a faculty member in Harvard Medical School's Department of Systems Biology, not only reconstructed the dynamics of memory, but also created a mathematical model that predicted how such a memory "device" might work.
"Synthetic biology is an incredibly exciting field, with more possibilities than many of us can imagine," says Silver, lead author of the paper to be published in the September 15 issue of the journal Genes and Development. "While this proof-of-concept experiment is simply one step forward, we've established a foundational technology that just might set the standard of what we should expect in subsequent work."
Like many emerging fields, there's still a bit of uncertainty over what, exactly, synthetic biology is. Ask any three scientists for a definition, and you'll probably get four answers.
Some see it as a means to boost the production of biotech products, such as proteins for pharmaceutical uses or other kinds of molecules for, say, environmental clean-up. Others see it as a means to creating computer platforms that may bypass many of the onerous stages of clinical trials. In such a scenario, a scientist would type the chemical structure of a drug candidate into a computer, and a program containing models of cellular metabolism could generate information on how people would react to that compound.
Either way, at it's core, synthetic biology boils down to gleaning insights into how biological systems work by reconstructing them. If you can build it, it forces you to understand it.
A team in Silver's Harvard Medical School lab led by Caroline Ajo-Franklin, now at Lawrence Berkeley National Laboratory, and postdoctoral scientist David Drubin decided to demonstrate that not only could they construct circuits out of genetic material, but they could also develop mathematical models whose predictive abilities match those of any electrical engineering system.
"That's the litmus test," says Drubin, "namely, building a biological device that does precisely what you predicted it would do."
The components of this memory loop were simple: two genes that coded for proteins called transcription factors.
Transcription factors regulate gene activity. Like a hand on a faucet, the transcription factor will grab onto a specific gene and control how much, or how little, of a particular protein the gene should make.
The researchers placed two of these newly synthesized, transcription factor-coding genes into a yeast cell, and then exposed the cell to galactose (a kind of sugar). The first gene, which was designed to switch on when exposed to galactose, created a transcription factor that grabbed on to, and thus activated, the second gene.
It was at this point that the feedback loop began.
The second gene also created a transcription factor. But this transcription factor, like a boomerang, swung back around and bound to that same gene from which it had originated, reactivating it. This caused the gene to once again create that very same transcription factor, which once again looped back and reactivated the gene.
In other words, the second gene continually switched itself on via the very transcription factor it created when it was switched on.
The researchers then eliminated the galactose, causing the first synthetic gene, the one that had initiated this whole process, to shut off. Even with this gene gone, the feedback loop continued.
"Essentially what happened is that the cell remembered that it had been exposed to galactose, and continued to pass this memory on to its descendents," says Ajo-Franklin. "So after many cell divisions, the feedback loop remained intact without galactose or any other sort of molecular trigger."
Most important, the entire construction of the device was guided by the mathematical model that the researchers developed.
"Think of how engineers build bridges," says Silver. "They design quantitative models to help them understand what sorts of pressure and weight the bridge can withstand, and then use these equations to improve the actual physical model. We really did the same thing. In fact, our mathematical model not only predicted exactly how our memory loop would work, but it informed how we synthesized the genes."
For synthetic biology, this kind of specificity is crucial. "If we ever want to create biological black boxes, that is, gene-based circuits like this one that you can plug into a cell and have it perform a specified task, we need levels of mathematical precision as exact as the kind that go into creating computer chips," she adds.
The researchers are now working to scale-up the memory device into a larger, more complex circuit, one that can, for example, respond to DNA damage in cells.
"One day we'd like to have a comprehensive library of these so-called black boxes," says Drubin. "In the same way you take a component off the shelf and plug it into a circuit and get a predicted reaction, that's what we'd one day like to do in cells."
For an animation of how to construct a synthetic memory loop see http://hms.harvard.edu/public/video/silver_illustration.mov
Full Citation: Caroline M. Ajo-Franklin(1), David A. Drubin(1), Julian A. Eskin(1), Elaine P.S. Gee(2), Dirk Landgraf(1), Ira Phillips(1), and Pamela A. Silver(1), "Rational design of memory in eukaryotic cells", Genes and Development, Volume 21, Issue 18: September 15, 2007
1-Department of Systems Biology, Harvard Medical School, 200 Longwood Ave., Boston MA 2-Harvard University Program in Biophysics, Massachusetts Institute of Technology, Cambridge, MA
Note: This story has been adapted from a news release issued by Harvard Medical School.

Fausto Intilla
www.oloscience.com

New Species May Form WIth A Little Help From Immune System

Source:

http://www.sciencedaily.com/releases/2007/09/070905103023.htm

Science Daily — Plant geneticists and animal breeders alike know the problem: single individuals or entire broods will not thrive, some die early, or remain, even if they survive, the runts of the litter and thus not useful for continued breeding programs. What is annoying for the breeder, fascinates geneticists and molecular biologists. The unfit offspring are an example that genetic material cannot always be combined at will.

Apparently there are reproductive barriers that not only prevent the exchange of genes between well established species, but also between varieties of one and the same species. How these barriers arise is of central importance if one wants to understand the origin of biodiversity. A research team led by Detlef Weigel from the Max Planck Institute of Developmental Biology in Germany and Jeff Dangl from the University of North Carolina has now shown that a mis-regulated immune system can establish reproductive barriers and might be a first step toward speciation. The international collaboration studied a genetic incompatibility known as hybrid necrosis, using thale cress, Arabidopsis thaliana.
The new work, reported in PLoS Biology, was based on the observation that unfit hybrids from different plant species are very similar. Their growth is retarded, the leaves become yellow and necrotic, the tissue collapses and they often do not survive to make flowers; the syndrome is generally known as hybrid necrosis. "We suspected that hybrid necrosis is always caused by the same biochemical mechanism," explains Weigel, director at the Max Planck Institute.
To test this hypothesis, the scientists took 280 genetically different strains of Arabidopsis from all over the world, which they crossed in 861 different combinations. Most of the hybrid plants were strong and grew normally, but 20 - or two percent - of the crosses produced only small necrotic and unhealthy plants. Genomics-based experiments showed that these hybrids all had a comparable profile of gene activity:
A common group of some 1000 genes were either more strongly or more weakly active in the hybrids than in their healthy parents. Moreover, this pattern was very similar to what is seen with a strong immune response mounted against pathogens during a normal infection. The plant immune response typically involves the sacrifice of a few cells at and around the infection site. But in the wimpy hybrids, healthy tissue also suffered - without pathogen infection. The hybrid plants apparently mistook their own cells for dangerous germs.
Although the genes that determined the abnormal autoimmunity were different in most crosses, the researchers discovered that often only two genes were required to cause the necrotic hybrid response. One of the fatal genes came from the father, the other from the mother. In one case that the researchers studied in more detail, they found that the gene that causes necrosis in hybrids, but not in the parents, is normally used to sense the presence of a pathogen.
The scientists emphasize, however, that the hybrids are not the victims of malfunctioning genes: in contrast to many hereditary diseases, the necrosis is not due to each parent carrying a defective copy of the same gene. Rather, there is a destructive interaction between two different genes, each of which evolved differently in the two parents. The genes on their own are harmless or even beneficial, since the parents are healthy. Only the combination of the altered gene variants creates problems. These types of genetic malfunction are often known as Dobzhanshy-Muller incompatibilities, after the two giants of early modern genetics who first studied these necrotic hybrids in fruit flies.
The results of the German-American team challenge the classical definition of a species, according to which individuals of one species can mate at will and produce fertile offspring. Apparently there are barriers to the free interchange of genes even within a species; after all, one out of 50 crosses in this study was not successful. "The formation of new species thus needs to be understood as a gradual process, where barriers within a species continually increase, until two groups cannot be crossed at all anymore" says Weigel.
While this view is widely accepted today, it is mostly unclear, why such genetic barriers arise in the first place. Which advantage has the plant, when sometimes all seeds from a cross die? The current study offers a possible explanation. The plant genome changes under pressure from pathogens.
"Plant and pathogen are locked in a race," says Dangl, professor and expert in the genetics of plant pathology at the University of North Carolina. The pathogens tirelessly develop new strategies to attack the plant and evade its immune system. The plant, in turn, tries to be prepared against as many new microbe "weapons" as possible. Armed to the teeth, it can happen that a harmless protein variant from a more distant relative is all of a sudden classified as dangerous and attacked.
The scientists are optimistic that their insights can be applied to other species. Common traits indicate that hybrid necrosis in crops such as wheat is caused by the same mechanisms as in tale cress. Dangl therefore believes that Arabidopsis can serve as a useful model for the understanding of hybrid necrosis in general. "Such a model would be very useful for breeding, since such genetic incompatibilities prevents some of the crosses breeders would like to make," according to Dangl.
The finding that only a few genes are responsible for each case of hybrid necrosis is particularly encouraging. It seems that only a few genetic changes are required to circumvent crossing barriers und to achieve a desired new combination of genetic traits. The flip side of the coin is that only minimal modifications in the genome can be sufficient to suppress the free exchange of genes between relatives, and that perhaps not much is needed to form a new species.
Authors of the study were Detlef Weigel, Kirsten Bomblies, Janne Lempe, Norman Warthmann and Christa Lanz from the Max Planck Institute for Developmental Biology, Tübingen, Germany; and Petra Epple und Jeffery L. Dangl from the University of North Carolina in Chapel Hill, North Carolina, USA.
Reference: Detlef Weigel, Kirsten Bomblies, Janne Lempe, Norman Warthmann, Christa Lanz and Petra Epple und Jeffery L. Dangl "Autoimmune response as a mechanism for a Dobzhansky-Muller-type incompatibility syndrome in plants" PLoS Biology 5, e236 (4. September 2007)
Note: This story has been adapted from a news release issued by Max-Planck-Gesellschaft.

Fausto Intilla

www.oloscience.com

In the Genome Race, the Sequel Is Personal

Source:

http://www.nytimes.com/2007/09/04/science/04vent.html?ref=science

By NICHOLAS WADE
Published: September 4, 2007

The race to decode the human genome may not be entirely over: the loser has come up with a new approach that may let him prevail in the end.

In 2003, a government-financed consortium of academic centers announced it had completed the human genome, fending off a determined challenge from the biologist J. Craig Venter. The consortium’s genome comprised just half the DNA contained in a normal cell, and the DNA used in the project came from a group of people from different racial and ethnic backgrounds.
But the loser in the race, Dr. Venter, could still have the last word. In a paper published today, his research team is announcing that it has decoded a new version of the human genome that some experts believe may be better than the consortium’s.
Called a full, or diploid genome, it consists of the DNA in both sets of chromosomes, one from each parent, and it is the normal genome possessed by almost all the body’s cells. And the genome the team has decoded belongs to just one person: Dr. Venter.
The new genome, Dr. Venter’s team reports, makes clear that the variation in the genetic programming carried by an individual is much greater than expected. In at least 44 percent of Dr. Venter’s genes, the copies inherited from his mother differ from those inherited from his father, according to the analysis published in Tuesday’s issue of PLoS Biology.
Huntington F. Willard, a geneticist at Duke University who has had early access to Dr. Venter’s genome sequence, said that the quality of the new genome was “exceptionally high” and that “until the next genome comes along this is the gold standard right now.”
Dr. Willard said it was “hugely better” than the consortium’s sequence, at least for his particular research interest.
“I don’t want to fan the fires but I like this, it’s a really good genome,” said Edward M. Rubin, a genome expert at the Lawrence Berkeley National Laboratory.
Dr. Venter’s race with the consortium began in 1998 when he spotted a quicker method of decoding the human genome. He tried to wrest this rich scientific prize from his academic rivals by co-founding a genome-decoding company called Celera. By June 2000, the two sides were neck and neck preparing a draft sequence of the genome. But in January 2002, Dr. Venter was abruptly fired as president of Celera. The consortium went on to claim victory when it announced its completion of the genome the next year.
But the consortium’s genome, though immensely useful to biologists, was full of gaps and only complete in the sense that it was the best that could be done with existing technology.
Dr. Venter has spent the last five years and an extra $10 million of his institute’s money in improving the draft genome he prepared at Celera. That genome was based mostly on his own DNA, and the new diploid version is entirely so. His critics may accuse him of an egocentricity of considerable dimension, but by analyzing his own genome he has sidestepped the problems of privacy and consent that could have arisen with other people’s DNA when he made the whole sequence publicly available, as he is doing now.
Like James Watson, the co-discoverer of DNA, whose genome is also being decoded, Dr. Venter believes strongly in making individual DNA sequences public to advance knowledge and hasten the era of personalized genomic medicine.
If other experts find that Dr. Venter’s genome is the best available, could it be said that he won the human genome race after all?
“There is this long history of Craig’s vanity, which for much of the scientific community is irritating,” Dr. Rubin said, declining to give a direct answer.
Asked the same question, Dr. Venter replied: “I’m not sure I’d want to be the one to say that, but we’re not through racing yet. I’ll let you know when we’ve stopped.”
James Shreeve, author of “The Genome War,” said, “I think he already believes he’s the true winner of the genome race for what he did at Celera,” noting that the consortium, too, believed it had won.
Though there are now novel technologies for decoding DNA very cheaply, Dr. Venter’s genome sequence could set a high bar for a long time. It was decoded with an old method, known as Sanger sequencing, that is expensive but analyzes stretches of DNA up to 800 units in length. The cheaper new technologies at present analyze pieces of DNA only 200 units or so long, and the shorter lengths are much harder to assemble into a complete genome.
Dr. Watson’s genome is being decoded with a next-generation machine developed by 454 Life Sciences. But the company’s researchers are putting the pieces in correct order by matching them to the consortium’s genome sequence rather than by doing an independent assembly.

Dr. Venter’s genome could be the gold standard for many years, especially if he continues to improve it. Samuel Levy, who led the J. Craig Venter Institute team that decoded the genome, said that it was a work in progress and that new versions would be published as the remaining gaps were closed. There are 4,500 gaps where the sequence of DNA units is uncertain, and no technology yet exists for decoding the large amounts of DNA at the center and tips of the chromosomes.

Biologists studying variation in the human genome, whether to discover causes of disease or for other reasons, have mostly looked at what are called SNPs or “snips,” which are sites on the genome where a single unit of DNA is changed.
But there are other kinds of variation, all of which can have consequences for a person. One type is called indels, where a single DNA unit has either been inserted or deleted from the genome. Another is copy number variation, in which the same gene can exist in multiple copies. There are also inversions, in which a stretch of DNA has been knocked out of its chromosome and reinserted the wrong way around. Dr. Venter’s genome has four million variations compared with the consortium’s, including three million snips, nearly a million indels and 90 inversions.
“This is the first time that anyone has had an accurate representation of how much variation there is in a human genome,” said Stephen W. Scherer of the University of Toronto, a co-author of the study.
Biologists had estimated that two individuals would be identical in 99.9 percent of their DNA, but the true figure now emerges as much less, around 99.5 percent, Dr. Scherer said.
The genome is being made publicly available on the database operated by the National Center for Biotechnology Information and is free for any use. Dr. Venter said he would add phenotypic information to the version on his own Web site, meaning medical records and other data to help researchers correlate his bodily characteristics with his DNA.
What little is understood about the human genome at present consists mostly of medical variants that put people at risk of disease. So interpreting a genome brings mostly adverse news. Dr. Venter reports that he has variants that increase his risk of alcoholism, coronary artery disease, obesity, Alzheimer’s disease, antisocial behavior and conduct disorder.
But these predictions are far from certain. As more individual genomes are decoded, the information from them will become more valuable, Dr. Venter said, provided that people can overcome “irrational fears of even seeing their genetic code.”
Although Dr. Venter has decoded the DNA sequence inherited from both of his parents, he does not yet know which sequences are from his mother and which from his father. The issue could be resolved by analyzing DNA from his mother, who is alive and well, and the matter is under consideration, Dr. Levy said. Dr. Venter has traced his ancestry for three generations and found that his mother’s and father’s ancestors came from England.
Next month, Dr. Venter will publish an autobiography, “A Life Decoded.” The book describes the twists and turns that led him down the unlikely path into scientific research. “Rebellious and disobedient,” as he describes himself, he dedicated his teenage years to the pursuit of young women and the California surf, to the detriment of his academic career.
He was drafted at the time of the Vietnam war and enlisted in the Navy. Because of a high I.Q. score, he was given a choice of any Navy career, from nuclear engineering to electronics. He chose the hospital corps school, because it was the only course that did not require any further enlistment. Only too late did he discover the reason. Corpsmen in Vietnam did not usually survive long enough to re-enlist — the half-life of medics in the field was six weeks, he writes.
Learning how to manipulate the Navy bureaucracy, he got himself assigned to the Navy hospital in Da Nang, where chances of survival were better. But the work was harrowing. He witnessed several hundred soldiers die on his operating table, mostly when he was massaging their heart or trying to breathe life into them.
“I learned more than any 20-year-old should ever have to about triage, about sorting those you can salvage from those you cannot do anything for except ease their pain as they died,” Dr. Venter writes in the autobiography.
He escaped from Vietnam with his life and an interest in medical research. With his lack of academic skills, this was a hard field for him to break into, but by 1975 he had a Ph.D. By the late 1980s, he was starting to make his mark as one of the few scientists who could get useful results out of the first DNA sequencing machines that were then becoming available.
He was the first to sequence the genome of a bacterium, Hemophilus influenzae, even though his grant application was turned down by the National Institutes of Health on the advice of experts who said his method would not work. With the human genome, an even greater prize, the pace of competition was intense, especially when his approach turned out to be more efficient than the one his rivals had chosen.
In the book, Dr. Venter says that detractors badmouthed his work, pressured other scientists not to cooperate with him and tried strenuously to block publication of his report, of which they had earlier maneuvered to be made co-authors.
“Like most human endeavors, science is driven in no small part by envy,” he writes.
Dr. Venter has never fully lost his youthful disrespect for authority and establishments. His investment in himself — choosing his own genome to sequence, naming his laboratory the J. Craig Venter Institute — may come across as vainglorious, but it can also be seen as a signal of survival, defying the establishments he believes have sought to crush him. However nettlesome he may seem to some of his colleagues, he has the charm and the personal skills to have recruited many highly able researchers to his teams.
Another reason for his success has been his skill at raising private finances to achieve research goals after being denied support from the National Institutes of Health. That a scientist of his ability has been forced to work outside the N.I.H.’s peer-review system puts peer review in a strange light. If his diploid human genome should become a standard, the success is one that he will have earned by perseverance and defiance of long odds.

Fausto Intilla

www.oloscience.com

A Genetic Trigger For The Cambrian Explosion Unraveled?

Source:

http://www.sciencedaily.com/releases/2007/08/070831180409.htm

Science Daily — A team of scientists led by young Croatian evolutionary geneticist Tomislav Domazet-Lošo from Ruder Boškovic Institute (RBI) in Zagreb, Croatia, developed a novel methodological approach in evolutionary studies. Using the method they named 'genomic phylostratigraphy', its authors shed new and unexpected light on some of the long standing macroevolutionary issues, which have been puzzling evolutionary biologists since Darwin.

The only direct method of research in evolutionary history involves analyzing the fossil remains of once living organisms, excavated in various localities throughout of the world. However, that approach often cannot provide the full evolutionary pathway of some species, as it requires uncovering of many fossils from various stages of its evolutionary history. As the fossil record is imperfect, the evolution research fundamentally hinges on luck factor in discovering the adequate paleontological sites.
However, the RBI team proposed a novel and interesting approach to bypass this obstacle. Namely, they suggested that the genome of every extant species carries the ‘snapshots’ of evolutionary epochs that species went trough. What's even more important, they also developed the method which enables evolution researchers to readily convert those individual 'snapshots’ into the full-length 'evolutionary movie' of a species.
Applying their new methodology on the fruit fly genomic data they tackled some of the most intriguing evolutionary puzzles - some of which distressed even Darwin himself. First, they demonstrated that parts of the living organism exposed to the environment – so called ‘ectoderm’ - are more prone to evolutionary changes. Further, they explained the evolutionary origin of the ‘germ layers’, the primary tissue forms that form during the first days after the conception of a new animal, and from which subsequently all other tissues are developed. Finally, they discovered the potential genetic trigger for the 'Cambrian explosion', a major global evolutionary event on the planet, when some 540 million years ago almost all animal forms known today suddenly 'appeared'.
The first public lecture on these findings will be given by dr. Domazet-Lošo on September 4th at 5. ISABS Conference in Forensic Genetics and Molecular Anthropology, held in Split, Croatia. The groundbreaking paper fully presenting the theory of genomic phylostratigraphy will appear in the November issue of 'Trends in genetics', the most established monthly journal in Genetics.
Reference: Domazet-Lošo, T. et al. (2007) 'A phylostratigraphy approach to uncover the genomic history of major adaptations in metazoan lineages'. Trends in Genetics (to appear in the November 2007 issue of the journal)
Note: This story has been adapted from a news release issued by Rudjer Boskovic Institute.

Fausto Intilla

www.oloscience.com