Chinese Medicine Yields Secrets: Atomic Mechanism of Two-Headed Molecule Derived from Chang Shan, a Traditional Chinese Herb

Chinese Medicine Yields Secrets: Atomic Mechanism of Two-Headed Molecule Derived from Chang Shan, a Traditional Chinese Herb

  9:49:00 AM    Science Lover

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The mysterious inner workings of Chang Shan -- a Chinese herbal medicine used for thousands of years to treat fevers associated with malaria -- have been uncovered thanks to a high-resolution structure solved at The Scripps Research Institute (TSRI).

Scripps Research Institute scientists have determined a molecular 
 structure that helps explain how the Chinese herbal medicine 
Chang Shan works. (Credit: Image courtesy of the Schimmel lab.)

Described in the journal Nature this week, the structure shows in atomic detail how a two-headed compound derived from the active ingredient in Chang Shan works. Scientists have known that this compound, called halofuginone (a derivative of the febrifugine), can suppress parts of the immune system -- but nobody knew exactly how.

The new structure shows that, like a wrench in the works, halofuginone jams the gears of a molecular machine that carries out "aminoacylation," a crucial biological process that allows organisms to synthesize the proteins they need to live. Chang Shan, also known as Dichroa febrifuga Lour, probably helps with malarial fevers because traces of a halofuginone-like chemical in the herb interfere with this same process in malaria parasites, killing them in an infected person's bloodstream.

"Our new results solved a mystery that has puzzled people about the mechanism of action of a medicine that has been used to treat fever from a malaria infection going back probably 2,000 years or more," said Paul Schimmel, PhD, the Ernest and Jean Hahn Professor and Chair of Molecular Biology and Chemistry and member of The Skaggs Institute for Chemical Biology at TSRI. Schimmel led the research with TSRI postdoctoral fellow Huihao Zhou, PhD.

Halofuginone has been in clinical trials for cancer, but the high-resolution picture of the molecule suggests it has a modularity that would make it useful as a template to create new drugs for numerous other diseases.

The Process of Aminoacylation and its Importance to Life

Aminoacylation is a crucial step in the synthesis of proteins, the end products of gene expression. When genes are expressed, their DNA sequence is first read and transcribed into RNA, a similar molecule. The RNA is then translated into proteins, which are chemically very different from DNA and RNA but are composed of chains of amino acid molecules strung together in the order called for in the DNA.

Necessary for this translation process are a set of molecules known as transfer RNAs (tRNAs), which shuttle amino acids to the growing protein chain where they are added like pearls on a string. But before the tRNAs can move the pearls in place, they must first grab hold of them.

Aminoacylation is the biological process whereby the amino acid's pearls are attached to these tRNA shuttles. A class of enzymes known as aminoacyl-tRNA synthetases is responsible for attaching the amino acids to the tRNAs, and Schimmel and his colleagues have been examining the molecular details of this process for years. Their work has given scientists insight into everything from early evolution to possible targets for future drug development.

Over time what has emerged as the picture of this process basically involves three molecular players: a tRNA, an amino acid and the aminoacyl-tRNA synthetase enzyme that brings them together. A fourth molecule called ATP is a microscopic form of fuel that gets consumed in the process.

The new work shows that halofuginone gets its potency by interfering with the tRNA synthetase enzyme that attaches the amino acid proline to the appropriate tRNA. It does this by blocking the active site of the enzyme where both the tRNA and the amino acid come together, with each half of the halofuginone blocking one side or the other.

Interestingly, said Schimmel, ATP is also needed for the halofuginone to bind. Nothing like that has ever been seen in biochemistry before.

"This is a remarkable example where a substrate of an enzyme (ATP) captures an inhibitor of the same enzyme, so that you have an enzyme-substrate-inhibitor complex," said Schimmel.

The article, "ATP-Directed Capture of Bioactive Herbal-Based Medicine on Human tRNA Synthetase," by Huihao Zhou, Litao Sun, Xiang-Lei Yang and Paul Schimmel was published in the journal Nature on Dec. 23, 2012.

This work was supported by the National Institutes of Health through grants #GM15539, #23562 and #88278 and by a fellowship from the National Foundation for Cancer Research.

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Strange Behavior: New Study Exposes Living Cells to Synthetic Protein

Strange Behavior: New Study Exposes Living Cells to Synthetic Protein

  10:22:00 AM    Science Lover

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One approach to understanding components in living organisms is to attempt to create them artificially, using principles of chemistry, engineering and genetics. A suite of powerful techniques -- collectively referred to as synthetic biology -- have been used to produce self-replicating molecules, artificial pathways in living systems and organisms bearing synthetic genomes.

The depletion of ATP in cells of the bacterium Escherichia coli causes them to transition to a filamentous state and form dense lipid structures known as endoliposomes. The structures can be clearly observed in these transmission electron micrographs of increasing magnification. (Credit: Image courtesy of Arizona State University)

In a new twist, John Chaput, a researcher at Arizona State University's Biodesign Institute and colleagues at the Department of Pharmacology, Midwestern University, Glendale, AZ have fabricated an artificial protein in the laboratory and examined the surprising ways living cells respond to it.

"If you take a protein that was created in a test tube and put it inside a cell, does it still function," Chaput asks. "Does the cell recognize it? Does the cell just chew it up and spit it out?" This unexplored area represents a new domain for synthetic biology and may ultimately lead to the development of novel therapeutic agents.

The research results, reported in the advanced online edition of the journal ACS Chemical Biology, describe a peculiar set of adaptations exhibited by Escherichia coli bacterial cells exposed to a synthetic protein, dubbed DX. Inside the cell, DX proteins bind with molecules of ATP, the energy source required by all biological entities.

"ATP is the energy currency of life," Chaput says. The phosphodiester bonds of ATP contain the energy necessary to drive reactions in living systems, giving up their stored energy when these bonds are chemically cleaved. The depletion of available intracellular ATP by DX binding disrupts normal metabolic activity in the cells, preventing them from dividing, (though they continue to grow).

After exposure to DX, the normally spherical E. coli bacteria develop into elongated filaments. Within the filamentous bacteria, dense intracellular lipid structures act to partition the cell at regular intervals along its length. These unusual structures, which the authors call endoliposomes, are an unprecedented phenomenon in such cells.

"Somewhere along the line of this filamentation, other processes begin to happen that we haven't fully understood at the genetic level, but we can see the results phenotypically," Chaput says. "These dense lipid structures are forming at very regular regions along the filamented cell and it looks like it could be a defense mechanism, allowing the cell to compartmentalize itself." This peculiar adaptation has never been observed in bacterial cells and appears unique for a single-celled organism.

Producing a synthetic protein like DX, which can mimic the elaborate folding characteristics of naturally occurring proteins and bind with a key metabolite like ATP is no easy task. As Chaput explains, a clever strategy known as mRNA display was used to produce, fine-tune and amplify synthetic proteins capable of binding ATP with high affinity and specificity, much as a naturally occurring ATP-binding protein would.

First, large libraries of random sequence peptides are formed from the four nucleic acids making up DNA, with each strand measuring around 80 nucleotides in length. These sequences are then transcribed into RNA with the help of an enzyme -- RNA polymerase. If a natural ribosome is then introduced, it attaches to the strand and reads the random sequence RNA as though it was a naturally-occurring RNA, generating a synthetic protein as it migrates along the strand. In this way, synthetic proteins based on random RNA sequences can be generated.

Exposing the batch of synthetic proteins to the target molecule and extracting those that bind can then select for ATP-binding proteins. But as Chaput explains, there's a problem: "The big question is how do you recover that genetic information? You can't reverse transcribe a protein back into DNA. You can't PCR amplify a protein. So we have to do all these molecular biology tricks."

The main trick involves an earlier step in the process. A molecular linker is chemically attached to the RNA templates, such that each RNA strand forms a bond with its newly translated protein. The mRNA-protein hybrids are exposed to selection targets (like ATP) over consecutive rounds of increasing stringency. After each round of selection, those library members that remain bound to the target are reverse-transcribed into cDNA (using their conveniently attached RNA messages), and then PCR amplified.

In the current study, E. coli cells exposed to DX transitioned into a filamentous form, which can occur naturally when such cells are subject to conditions of stress. The cells display low metabolic activity and limited cell division, presumably owing to their ATP-starved condition.

The study also examined the ability of E. coli to recover following DX exposure. The cells were found to enter a quiescent state known as viable but non-culturable (VBNC), meaning that they survived ATP sequestration and returned to their non-filamentous state after 48 hours, but lost their reproductive capacity. Further, this condition was difficult to reverse and seems to involve a fundamental reprogramming of the cell.

In an additional response to DX, the filamentous cells form previously undocumented structures, which the authors refer to as endoliposomes. These dense lipid concentrations, spanning the full width of the filamented E. coli, segment the cells into distinct compartments, giving the cells a stringbean-like appearance under the microscope.

The authors speculate that this adaptation may be an effort to maintain homeostasis in regions of the filamentous cell, which have essentially been walled off from the intrusion of ATP-depleting DX. They liken endoliposomes to the series of water-tight compartments found in submarines which are used to isolate damaged sections of the ship and speculate that DX-exposed cells are partitioning their genetic information into regions where it can be safely quarantined. Such self-compartmentalization is known to occur in some eukaryotic cells, but has not been previously observed in prokaryotes like E. coli.

The research indicates that there is still a great deal to learn about bacterial behavior and the repertoire of responses available when such cells encounter novel situations, such as an unfamiliar, synthetic protein. The study also notes that many infectious agents rely on a dormant state, (similar to the VBNC condition observed in the DX-exposed E. coli), to elude detection by antibiotics. A better understanding of the mechanisms driving this behavior could provide a new approach to targeting such pathogens.

The relative safety of E. coli as a model organism for study may provide a fruitful tool for more in-depth investigation of VBNC states in pathogenic organisms. Further, given ATP's central importance for living organisms, its suppression may provide another avenue for combating disease. One example would be an engineered bacteriophage capable of delivering DX genes to pathogenic organisms.

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Researchers Watch Tiny Living Machines Self-Assemble

Researchers Watch Tiny Living Machines Self-Assemble

  1:05:00 PM    Science Lover

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Enabling bioengineers to design new molecular machines for nanotechnology applications is one of the possible outcomes of a study by University of Montreal researchers that was published in Nature Structural and Molecular Biology June 10. The scientists have developed a new approach to visualize how proteins assemble, which may also significantly aid our understanding of diseases such as Alzheimer's and Parkinson's, which are caused by errors in assembly.

Vallée-Bélisle and Michnick have developed a new 
approach to visualize how proteins assemble, which may 
also significantly aid our understanding of diseases such 
as Alzheimer’s and Parkinson’s, which are caused by errors 
in assembly. Here shown are two different assembly stages 
(purple and red) of the protein ubiquitin and the fluorescent 
probe used to visualize these stage (tryptophan: see yellow).

"In order to survive, all creatures, from bacteria to humans, monitor and transform their environments using small protein nanomachines made of thousands of atoms," explained the senior author of the study, Prof. Stephen Michnick of the university's department of biochemistry. "For example, in our sinuses, there are complex receptor proteins that are activated in the presence of different odor molecules. Some of those scents warn us of danger; others tell us that food is nearby." Proteins are made of long linear chains of amino acids, which have evolved over millions of years to self-assemble extremely rapidly -- often within thousandths of a split second -- into a working nanomachine. "One of the main challenges for biochemists is to understand how these linear chains assemble into their correct structure given an astronomically large number of other possible forms," Michnick said.

"To understand how a protein goes from a linear chain to a unique assembled structure, we need to capture snapshots of its shape at each stage of assembly said Dr. Alexis Vallée-Bélisle, first author of the study. "The problem is that each step exists for a fleetingly short time and no available technique enables us to obtain precise structural information on these states within such a small time frame. We developed a strategy to monitor protein assembly by integrating fluorescent probes throughout the linear protein chain so that we could detect the structure of each stage of protein assembly, step by step to its final structure."

The protein assembly process is not the end of its journey, as a protein can change, through chemical modifications or with age, to take on different forms and functions. "Understanding how a protein goes from being one thing to becoming another is the first step towards understanding and designing protein nanomachines for biotechnologies such as medical and environmental diagnostic sensors, drug synthesis of delivery," Vallée-Bélisle said.

This research was supported by the Natural Sciences and Engineering Research Council of Canada and Le fond de recherché du Québec, Nature et Technologie. The article, "Visualizing transient protein folding intermediates by tryptophan scanning mutagenesis," published in Nature Structural & Molecular Biology, was coauthored by Alexis Vallée-Bélisle and Stephen W. Michnick of the Département de Biochimie de l'Université de Montréal. The University of Montreal is known officially as Université de Montréal.

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Engineers Reverse E. Coli Metabolism for Quick Production of Fuels, Chemicals

Engineers Reverse E. Coli Metabolism for Quick Production of Fuels, Chemicals

  10:54:00 PM    Science Lover

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In a biotechnological tour de force, Rice University engineering researchers this week unveiled a new method for rapidly converting simple glucose into biofuels and petrochemical substitutes. In a paper published online in Nature, Rice's team described how it reversed one of the most efficient of all metabolic pathways -- the beta oxidation cycle -- to engineer bacteria that produce biofuel at a breakneck pace.

Rice University engineering researchers Ramon Gonzalez (left) and
Clementina Dellomonaco reversed one of the most efficient of all
metabolic pathways -- the beta oxidation cycle -- to engineer bacteria
that make biofuels at a breakneck pace. (Credit: Jeff Fitlow/Rice
University)

Just how fast are Rice's single-celled chemical factories? On a cell-per-cell basis, the bacteria produced the butanol, a biofuel that can be substituted for gasoline in most engines, about 10 times faster than any previously reported organism.


"That's really not even a fair comparison because the other organisms used an expensive, enriched feedstock, and we used the cheapest thing you can imagine, just glucose and mineral salts," said Ramon Gonzalez, associate professor of chemical and biomolecular engineering at Rice and lead co-author of the Nature study.


Gonzalez's laboratory is in a race with hundreds of labs around the world to find green methods for producing chemicals like butanol that have historically come from petroleum.


"We call these 'drop-in' fuels and chemicals, because their structure and properties are very similar, sometimes identical, to petroleum-based products," he said. "That means they can be 'dropped in,' or substituted, for products that are produced today by the petrochemical industry."




Butanol is a relatively short molecule, with a backbone of just four carbon atoms. Molecules with longer carbon chains have been even more troublesome for biotech producers to make, particularly molecules with chains of 10 or more carbon atoms. Gonzalez said that's partly because researchers have focused on ramping up the natural metabolic processes that cells use to build long-chain fatty acids. Gonzalez and students Clementina Dellomonaco, James Clomburg and Elliot Miller took a completely different approach.


"Rather than going with the process nature uses to build fatty acids, we reversed the process that it uses to break them apart," Gonzalez said. "It's definitely unconventional, but it makes sense because the routes nature has selected to build fatty acids are very inefficient compared with the reversal of the route it uses to break them apart."


The beta oxidation process is one of biology's most fundamental, Gonzalez said. Species ranging from single-celled bacteria to human beings use beta oxidation to break down fatty acids and generate energy.


In the Nature study, Gonzalez's team reversed the beta oxidation cycle by selectively manipulating about a dozen genes in the bacteria Escherichia coli. They also showed that selective manipulations of particular genes could be used to produce fatty acids of particular lengths, including long-chain molecules like stearic acid and palmitic acid, which have chains of more than a dozen carbon atoms.


"This is not a one-trick pony," Gonzalez said. "We can make many kinds of specialized molecules for many different markets. We can also do this in any organism. Some producers prefer to use industrial organisms other than E. coli, like algae or yeast. That's another advantage of using reverse-beta oxidation, because the pathway is present in almost every organism."


The research was funded by Rice University.

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Editing the Genome: Scientists Unveil New Tools for Rewriting the Code of Life

Editing the Genome: Scientists Unveil New Tools for Rewriting the Code of Life

  2:50:00 AM    Science Lover

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The power to edit genes is as revolutionary, immediately useful and unlimited in its potential as was Johannes Gutenberg's printing press. And like Gutenberg's invention, most DNA editing tools are slow, expensive, and hard to use -- a brilliant technology in its infancy. Now, Harvard researchers developing genome-scale editing tools as fast and easy as word processing have rewritten the genome of living cells using the genetic equivalent of search and replace -- and combined those rewrites in novel cell strains, strikingly different from their forebears.

Researchers have unveiled genome-engineering technologies capable of fundamentally re-engineering genomes from the nucleotide to the megabase scale. Treating the chromosome as both an editable and an evolvable template, the researchers have demonstrated methods to rewrite a cell's genome through powerful new tools for biotechnology, energy and agriculture. (Credit: iStockphoto/Zmeel Photography)

"The payoff doesn't really come from making a copy of something that already exists," said George Church, a professor of genetics at Harvard Medical School who led the research effort in collaboration with Joe Jacobson, an associate professor at the Media Lab at the Massachusetts Institute of Technology. "You have to change it -- functionally and radically."

Such change, Church said, serves three goals. The first is to add functionality to a cell by encoding for useful new amino acids. The second is to introduce safeguards that prevent cross-contamination between modified organisms and the wild. A third, related aim, is to establish multi-viral resistance by rewriting code hijacked by viruses. In industries that cultivate bacteria, including pharmaceuticals and energy, such viruses affect up to 20 percent of cultures. A notable example afflicted the biotech company Genzyme, where estimates of losses due to viral contamination range from a few hundred million dollars to more than $1 billion.

In a paper scheduled for publication July 15 in Science, the researchers describe how they replaced instances of a codon -- a DNA "word" of three nucleotide letters -- in 32 strains of E. coli, and then coaxed those partially-edited strains along an evolutionary path toward a single cell line in which all 314 instances of the codon had been replaced. That many edits surpasses current methods by two orders of magnitude, said Harris Wang, a research fellow in Church's lab at the Wyss Institute for Biologically Inspired Engineering who shares lead-author credit on the paper with Farren Isaacs, an assistant professor of molecular, cellular and developmental biology at Yale University and former Harvard research fellow, and Peter Carr, a research scientist at the MIT Media Lab.



In the genetic code, most codons specify an amino acid, a protein building block. But a few codons tell the cell when to stop adding amino acids to a protein chain, and it was one of these "stop" codons that the Harvard researchers targeted. With just 314 occurrences, the TAG stop codon is the rarest word in the E. coli genome, making it a prime target for replacement. Using a platform called multiplex automated genome engineering, or MAGE, the team replaced instances of the TAG codon with another stop codon, TAA, in living E. coli cells. (Unveiled by the team in 2009, the MAGE process has been called an evolution machine for its ability to accelerate targeted genetic change in living cells.)

While MAGE, a small-scale engineering process, yielded cells in which TAA codons replaced some but not all TAG codons, the team constructed 32 strains that, taken together, included every possible TAA replacement. Then, using bacteria's innate ability to trade genes through a process called conjugation, the researchers induced the cells to transfer genes containing TAA codons at increasingly larger scales. The new method, called conjugative assembly genome engineering, or CAGE, resembles a playoff bracket -- a hierarchy that winnows 16 pairs to eight to four to two to one -- with each round's winner possessing more TAA codons and fewer TAG, explains Isaacs, who invokes "March Madness."

"We're testing decades-old theories on the conservation of the genetic code," Isaacs said. "And we're showing on a genome-wide scale that we're able to make these changes."

Eager to share their enabling technology, the team published their results as CAGE reached the semifinal round. Results suggested that the final four strains were healthy, even as the team assembled four groups of 80 engineered alterations into stretches of the chromosome surpassing 1 million DNA base pairs. "We encountered a great deal of skepticism early on that we could make so many changes and preserve the health of these cells," Carr said. "But that's what we've seen."

The researchers are confident that they will create a single strain in which TAG codons are completely eliminated. The next step, they say, is to delete the cell's machinery that reads the TAG gene -- freeing up the codon for a completely new purpose, such as encoding a novel amino acid.

"We're trying to challenge people," Wang said, "to think about the genome as something that's highly malleable, highly editable."

This research was funded by U.S. Department of Energy and the National Science Foundation.

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Ancient Viral Invasion Shaped Human Genome

Ancient Viral Invasion Shaped Human Genome

  2:14:00 AM    Science Lover

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Scientists at the Genome Institute of Singapore (GIS), a biomedical research institute of the Agency for Science, Technology and Research (A*STAR), and their colleagues from the National University of Singapore, Nanyang Technological University, Duke-NUS Graduate Medical School and Princeton University have recently discovered that viruses that 'invaded' the human genome millions of years ago have changed the way genes get turned on and off in human embryonic stem (ES) cells.

Scientists have discovered that viruses that "invaded" 
the human genome millions of years ago have changed the 
way genes get turned on and off in human embryonic
stem cells. (Credit: iStockphoto/Martin McCarthy)

The study provides definitive proof of a theory that was first proposed in the 1950s by Nobel Laureate in physiology and medicine, Barbara McClintock, who hypothesized that transposable elements, mobile pieces of the genetic material (DNA), such as viral sequences, could be "control elements" that affect gene regulation once inserted in the genome.

This finding is an important contribution to the advancement of stem cell research and to its potential for regenerative medicine. Led by GIS Senior Group Leader Dr Guillaume Bourque, the study was published in Nature Genetics on June 6, 2010.

Through the use of new sequencing technologies, the scientists studied the genomic locations of three regulatory proteins (OCT4, NANOG and CTCF) in human and mouse embryonic stem (ES) cells. Interestingly, while the scientists found a lot of similarities, they also found many differences in the methods and the types of genes that are being regulated in humans. In particular, it was discovered that specific types of viruses that inserted themselves in the human genomes millions of years ago have dramatically changed the gene regulatory network in human stem cells.

"This study is a computational and experimental tour de force. It provides undeniable evidence that some transposable elements, which are too often dismissed as merely junk DNA, are key components of a regulatory code underlying human development," said Dr Cedric Feschotte, Associate Professor of the University of Texas Arlington.

The comparisons between the human and mouse model system in the study of gene regulatory networks help to advance the understanding of how stem cells differentiate into various cell types of the body. "This understanding is crucial in the improved development of regenerative medicine for diseases such as Parkinson's disease and leukaemia," said Dr Bourque. "Despite the advantages of using mouse ES cells in the study of gene regulatory networks, further research must focus more directly on human stem cells. This is due to the inherent challenges of converting the results of studies done from one species to that of the next. More research will need to be done in both human and non-human primate stem cells for findings on stem cells to be used in clinical application."

Prof Raymond L. White, PhD, Rudi Schmid Distinguished Professor of Neurology, University of California said, "The paper reports very exciting new findings that establish a new and fundamentally distinct mechanism for the regulation of gene expression. By comparing the genomes of mouse with human, the scientists were able to show that the binding sites for gene regulatory factors are very often not in the same place between the two species. This by itself would be very surprising, but the investigators go further and demonstrate that many of the sites are imbedded within a class of DNA sequences called "transposable" elements because of their ability to move to new places in the genome. There are a number of such elements believed to be the evolutionary remnants of viral genomes, but it was very surprising to learn that they were carrying binding sites for regulatory elements to new locations. These changes in regulation would be expected to create major changes in the organisms which carry them. Indeed, many think that regulatory changes are at the heart of speciation and may have played a large role in the evolution of humans from their predecessors. This is likely to be a landmark paper in the field."

Dr Eddy Rubin, Director of the U.S. Department of Energy Joint Genome Institute and Director of the Genomics Division at Lawrence Berkeley National Laboratory in Berkeley added, "This study using a comparative genomics strategy discovered important human specific properties of the regulatory network in human ES cells. This information is significant and should contribute to helping move the regenerative medicine field forward."

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Mitochondrial Eve: Mother of All Humans Lived 200,000 Years Ago

Mitochondrial Eve: Mother of All Humans Lived 200,000 Years Ago

  10:05:00 AM    Science Lover

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The most robust statistical examination to date of our species' genetic links to "mitochondrial Eve" -- the maternal ancestor of all living humans -- confirms that she lived about 200,000 years ago. The Rice University study was based on a side-by-side comparison of 10 human genetic models that each aim to determine when Eve lived using a very different set of assumptions about the way humans migrated, expanded and spread across Earth.

Artist's cross section of a mitochondrion. (Credit: iStockphoto/David Marchal)

The research is available online in the journal Theoretical Population Biology.

"Our findings underscore the importance of taking into account the random nature of population processes like growth and extinction," said study co-author Marek Kimmel, professor of statistics at Rice. "Classical, deterministic models, including several that have previously been applied to the dating of mitochondrial Eve, do not fully account for these random processes."

The quest to date mitochondrial Eve (mtEve) is an example of the way scientists probe the genetic past to learn more about mutation, selection and other genetic processes that play key roles in disease.

"This is why we are interested in patterns of genetic variability in general," Kimmel said. "They are very important for medicine."

For example, the way scientists attempt to date mtEve relies on modern genetic techniques. Genetic profiles of random blood donors are compared, and based upon the likenesses and differences between particular genes, scientists can assign a number that describes the degree to which any two donors are related to one another.

Using mitochondrial genomes to gauge relatedness is a way for geneticists to simplify the task of finding common ancestors that lived long ago. That is because the entire human genome contains more than 20,000 genes, and comparing the differences among so many genes for distant relatives is problematic, even with today's largest and fastest supercomputers.

But mitochondria -- the tiny organelles that serve as energy factories inside all human cells -- have their own genome. Besides containing 37 genes that rarely change, they contain a "hypervariable" region, which changes fast enough to provide a molecular clock calibrated to times comparable to the age of modern humanity. Because each person's mitochondrial genome is inherited from his or her mother, all mitochondrial lineages are maternal.

To infer mtEve's age, scientists must convert the measures of relatedness between random blood donors into a measure of time.

"You have to translate the differences between gene sequences into how they evolved in time," said co-author Krzysztof Cyran, vice head of the Institute of Informatics at Silesian University of Technology in Gliwice, Poland. "And how they evolved in time depends upon the model of evolution that you use. So, for instance, what is the rate of genetic mutation, and is that rate of change uniform in time? And what about the process of random loss of genetic variants, which we call genetic drift?"

Within each model, the answers to these questions take the form of coefficients -- numeric constants that are plugged into the equation that returns the answer for when mtEve lived.

Each model has its own assumptions, and each assumption has mathematical implications. To further complicate matters, some of the assumptions are not valid for human populations. For example, some models assume that population size never changes. That is not true for humans, whose population has grown exponentially for at least several thousand generations. Other models assume perfect mixing of genes, meaning that any two humans anywhere in the world have an equal chance of producing offspring.

Cyran said human genetic models have become more complex over the past couple of decades as theorists have tried to correct for invalid assumptions. But some of the corrections -- like adding branching processes that attempt to capture the dynamics of population growth in early human migrations -- are extremely complex. Which raises the question of whether less complex models might do equally well in capturing what's occurring.

"We wanted to see how sensitive the estimates were to the assumptions of the models," Kimmel said. "We found that all of the models that accounted for random population size -- such as different branching processes -- gave similar estimates. This is reassuring, because it shows that refining the assumptions of the model, beyond a certain point, may not be that important in the big picture."

The research was supported by grants from the Polish Ministry of Science and Higher Education and the Cancer Prevention and Research Institute of Texas. It has resulted from a standing collaboration between Rice University and Silesian University of Technology.

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Evolutionary Surprise: Eight Percent of Human Genetic Material Comes from a Virus

Evolutionary Surprise: Eight Percent of Human Genetic Material Comes from a Virus

  10:15:00 PM    Science Lover

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About eight percent of human genetic material comes from a virus and not from our ancestors, according to researchers in Japan and the U.S.

A new study shows that the genomes of humans and other mammals contain DNA derived from the insertion of bornaviruses, RNA viruses whose replication and transcription takes place in the nucleus. (Credit: iStockphoto)

The study, and an accompanying News & Views article by University of Texas at Arlington biology professor Cédric Feschotte, is published in the journal Nature.

The research showed that the genomes of humans and other mammals contain DNA derived from the insertion of bornaviruses, RNA viruses whose replication and transcription takes place in the nucleus. Feschotte wrote on recent research led by Professor Keizo Tomonaga at Osaka University in Japan. Feschotte said this virally transmitted DNA may be a cause of mutation and psychiatric disorders such as schizophrenia and mood disorders.

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Evolution's Footprints in Human Genome Precisely Tracked Using New Approach

Evolution's Footprints in Human Genome Precisely Tracked Using New Approach

  10:08:00 AM    Science Lover

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Fossils may provide tantalizing clues to human history but they also lack some vital information, such as revealing which pieces of human DNA have been favored by evolution because they confer beneficial traits -- resistance to infection or the ability to digest milk, for example. These signs can only be revealed through genetic studies of modern humans and other related species, though the task has proven difficult.

Researchers have developed a new method for pinpointing pieces of human DNA have been favored by evolution within the human genome. The technique offers greater precision and resolution than ever before. (Credit: iStockphoto)

Now, in a paper appearing in the January 7 edition of Science Express, researchers describe a method for pinpointing these preferred regions within the human genome that offers greater precision and resolution than ever before, and the possibility of deeply understanding both our genetic past and present.

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First Genetic Map of Han Chinese May Aid Search for Disease Susceptibility Genes

First Genetic Map of Han Chinese May Aid Search for Disease Susceptibility Genes

  1:30:00 AM    Science Lover

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The first genetic historical map of the Han Chinese, the largest ethnic population in the world, as they migrated from south to north over evolutionary time, was published online November 25 in the American Journal of Human Genetics by scientists at the Genome Institute of Singapore (GIS).

DNA on abstract background. Researchers have published the first genetic historical map of the Han Chinese, the largest ethnic population in the world, as they migrated from south to north over evolutionary time. (Credit: iStockphoto/Andrey Prokhorov)

Based on genome-wide DNA variation information in over 6,000 Han Chinese samples from 10 provinces in China, this new map provides information about the population structure and evolutionary history of this group of people that can help scientists to identify subtle differences in the genetic diversity of Asian populations.

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Inefficient Selection: New Evolutionary Mechanism Accounts For Some Of Human Biological Complexity

Inefficient Selection: New Evolutionary Mechanism Accounts For Some Of Human Biological Complexity

  10:29:00 PM    Science Lover

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A painstaking analysis of thousands of genes and the proteins they encode shows that human beings are biologically complex, at least in part, because of the way humans evolved to cope with redundancies arising from duplicate genes.

Genomic and proteomic analysis has found a new evolutionary mechanism that accounts for some of the biological complexity of human beings. (Credit: iStockphoto/Liang Zhang)

"We have found a specific evolutionary mechanism to account for a portion of the intricate biological complexity of our species," said Ariel Fernandez, professor of bioengineering at Rice University. "It is a coping mechanism, a process that enables us to deal with the fitness consequences of inefficient selection. It enables some of our proteins to become more specialized over time, and in turn makes us more complex."

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The Story Of X: Evolution Of A Sex Chromosome

The Story Of X: Evolution Of A Sex Chromosome

  1:06:00 AM    Science Lover

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Move over, Y chromosome – it's time X got some attention.

In the first evolutionary study of the chromosome associated with being female, University of California, Berkeley, biologist Doris Bachtrog and her colleagues show that the history of the X chromosome is every bit as interesting as the much-studied, male-determining Y chromosome, and offers important clues to the origins and benefits of sexual reproduction.

"Contrary to the traditional view of being a passive player, the X chromosome has a very active role in the evolutionary process of sex chromosome differentiation," said Bachtrog, an assistant professor of integrative biology and a member of UC Berkeley's Center for Theoretical Evolutionary Genomics.

Bachtrog, UC Berkeley post-doctoral fellow Jeffrey D. Jensen and former UC San Diego post-doc Zhi Zhang, now at the University of Munich, detail their findings in this week's edition of the open-access journal PLoS Biology.

"In our manuscript, we demonstrate for the first time the flip side of the sex chromosome evolution puzzle: The X chromosome undergoes periods of intense adaptation in the evolutionary process of creating new sections of the genome that govern sexual differentiation in many species, including our own," she said.

Not all animals and plants employ genes to determine if an embryo becomes male or female. Many reptiles, for example, rely on environmental cues such as temperature to specify male or female.

But in life forms that do set aside a pair of chromosomes to specify sex – from fruit flies to mammals and some plants – the two X chromosomes inherited by females look nearly identical to the other non-sex chromosomes, so-called autosomes, Bachtrog said. The Y chromosome, however, which is inherited by males in concert with one X chromosome, is a withered version of the X, having lost many genes since it stopped recombining with the X chromosome.

In mammals, that probably took place about 150 million years ago, while in the fruit fly Drosophila melanogaster, a laboratory favorite, the sex chromosomes arose independently about 100 million years ago. In both humans and fruit flies, the Y chromosome has dwindled from a few thousand genes to a few dozen.

Hence the intense interest in why and how the Y chromosome lost genes once it stopped interacting with the X. Scientists have found that, as the only chromosome pair that doesn't break and recombine every time a cell divides, the XY pair in males is unable to take advantage of the main way deleterious genetic mutations are eliminated. The XX pair in females does recombine, but for the Y, the only way to get rid of a bad mutation in a gene is to inactivate or delete the entire gene. Over millions of years, inactive genes are lost, and the Y shrinks.

"If you have no recombination, natural selection is less effective at removing detrimental genes," said Bachtrog. "Y is an asexual chromosome, and it pays a price for that: It keeps losing genes."

Bachtrog, whose career has revolved mostly around the study of the degeneration of the Y chromosome, decided to focus on the X chromosome several years ago and went about searching for sex chromosome pairs that have arisen more recently – and thus might be in the process of adapting to their new role. Her paper centers around study of the three sex chromosomes in a rare western fruit fly, Drosophila miranda, a darker-colored cousin of D. melanogaster. (Many creatures have more than one pair of sex chromosomes; the platypus, for example, has five pairs, all inherited together.)

While one of D. miranda's sex chromosomes is descended from the original sex chromosome that appeared in Drosophila nearly 100 million years ago, a second originated perhaps 10 million years ago, and the third about a million years ago. The older two look much alike, Bachtrog said: The Y chromosome in each pair has lost genes to become a shadow of its former self, while the two X chromosomes are indistinguishable from each other.

The third and youngest sex chromosome is different. The Y is not yet shriveled, though it contains many non-functional genes – about half the total – that will eventually be lost. The X, which is dubbed neo-X, is undergoing rapid change, however, with about 10 times the normal amount of adaptation seen in the autosomes, according to the researchers.

By adaptation, Bachtrog means that the gene sequences in the X chromosome are becoming fixed as random mutations have finally settled on a few beneficial changes that accommodate the increasingly irrelevant Y chromosome. Between 10 and 15 percent of neo-X genes show adaptation, compared to only 1-3 percent of autosome genes.

"In hindsight, that is not surprising," Bachtrog said. "Neo-X is facing a much more challenging situation than the autosomes because its pair, the Y chromosome, is degenerating. Its genes are no longer producing proteins, so neo-X has to compensate by up-regulating its genes. We find a lot of genes on the X chromosome are involved in dosage compensation."

In humans, for example, all genes on the X chromosome are twice as active to account for the lack of genes on the Y. Women accommodate this by inactivating one entire X chromosome so as not to produce too much protein, Bachtrog said.

Another change in neo-X that Bachtrog suspects is taking place is the elimination of genes that are harmful to females. Biologists have realized recently that some genes have opposite effects in males and females, and evolution is a tug of war between males jettisoning genes that they find detrimental only to have females put them back, and vice versa.

"A good place to put sexually antagonistic genes that are beneficial to one sex but detrimental to the other is on the sex chromosomes," she said. The Y always ends up in the male, she said, so genes on the Y chromosome won't affect females.

"Conversely, the X chromosome becomes feminized with genes that are good for the female but detrimental to the male," said Bachtrog, adding that the X also becomes demasculinized, losing genes that are of use only in the male.

In search of more insights into the evolution of the X chromosome, Bachtrog said she is looking for fruit fly species with older and younger sex chromosomes "to study sex chromosome evolution in action." She said evidence suggests that adaptation to being a sex chromosome is most intense between 1 and 10 million years after it starts. Bachtrog also is completing assembly of the genome sequence for D. miranda, which is not among the 12 species of Drosophila currently targeted by the genome sequencing community. She hopes that the fly will become a model system like D. melanogaster.

"Now, finally, we are within reach of studying model systems like D. miranda that we couldn't think of several years ago," she said, predicting that "whole genome comparisons will revolutionize evolutionary biology, ecology and many other fields."

The research was funded by the National Institutes of Health, an Alfred P. Sloan Faculty Research Fellowship in Molecular and Computational Biology and a David and Lucile Packard Foundation Fellowship.

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New Nucleotide In DNA Could Revolutionize Epigenetics

New Nucleotide In DNA Could Revolutionize Epigenetics

  8:13:00 PM    Science Lover

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Chemical structure of cytosine, one of the four nucleotide bases that make up DNA.
New research shows that two additional nucleotides -- 5-methylcytosine and 5-hydroxymethylcytosine -- can sometimes replace cytosine in the DNA
double helix to regulate which genes are expressed. (Credit: Wikimedia Commons)

Anyone who studied a little genetics in high school has heard of adenine, thymine, guanine and cytosine – the A, T, G and C that make up the DNA code. But those are not the whole story. The rise of epigenetics in the past decade has drawn attention to a fifth nucleotide, 5-methylcytosine (5-mC), that sometimes replaces cytosine in the famous DNA double helix to regulate which genes are expressed. And now there's a sixth: 5-hydroxymethylcytosine.

In experiments to be published online April 16 by Science, researchers reveal an additional character in the mammalian DNA code, opening an entirely new front in epigenetic research.

The work, conducted in Nathaniel Heintz's Laboratory of Molecular Biology at The Rockefeller University, suggests that a new layer of complexity exists between our basic genetic blueprints and the creatures that grow out of them. "This is another mechanism for regulation of gene expression and nuclear structure that no one has had any insight into," says Heintz, who is also a Howard Hughes Medical Institute investigator. "The results are discrete and crystalline and clear; there is no uncertainty. I think this finding will electrify the field of epigenetics."

Genes alone cannot explain the vast differences in complexity among worms, mice, monkeys and humans, all of which have roughly the same amount of genetic material. Scientists have found that these differences arise in part from the dynamic regulation of gene expression rather than the genes themselves. Epigenetics, a relatively young and very hot field in biology, is the study of nongenetic factors that manage this regulation.

One key epigenetic player is DNA methylation, which targets sites where cytosine precedes guanine in the DNA code. An enzyme called DNA methyltransferase affixes a methyl group to cytosine, creating a different but stable nucleotide called 5-methylcytosine. This modification in the promoter region of a gene results in gene silencing.

Some regional DNA methylation occurs in the earliest stages of life, influencing differentiation of embryonic stem cells into the different cell types that constitute the diverse organs, tissues and systems of the body. Recent research has shown, however, that environmental factors and experiences, such as the type of care a rat pup receives from its mother, can also result in methylation patterns and corresponding behaviors that are heritable for several generations. Thousands of scientific papers have focused on the role of 5-methylcytosine in development.

The discovery of a new nucleotide may make biologists rethink their approaches to investigating DNA methylation. Ironically, the latest addition to the DNA vocabulary was found by chance during investigations of the level of 5-methylcytosine in the very large nuclei of Purkinje cells, says Skirmantas Kriaucionis, a postdoctoral associate in the Heintz lab, who did the research. "We didn't go looking for this modification," he says. "We just found it."

Kriaucionis was working to compare the levels of 5-methylcytosine in two very different but connected neurons in the mouse brain — Purkinje cells, the largest brain cells, and granule cells, the most numerous and among the smallest. Together, these two types of cells coordinate motor function in the cerebellum. After developing a new method to separate the nuclei of individual cell types from one another, Kriaucionis was analyzing the epigenetic makeup of the cells when he came across substantial amounts of an unexpected and anomalous nucleotide, which he labeled 'x.'

It accounted for roughly 40 percent of the methylated cytosine in Purkinje cells and 10 percent in granule neurons. He then performed a series of tests on 'x,' including mass spectrometry, which determines the elemental components of molecules by breaking them down into their constituent parts, charging the particles and measuring their mass-to-charge ratio. He repeated the experiments more than 10 times and came up with the same result: x was 5-hydroxymethylcytosine, a stable nucleotide previously observed only in the simplest of life forms, bacterial viruses. A number of other tests showed that 'x' could not be a byproduct of age, DNA damage during the cell-type isolation procedure or RNA contamination. "It's stable and it's abundant in the mouse and human brain," Kriaucionis says. "It's really exciting."

What this nucleotide does is not yet clear. Initial tests suggested that it may play a role in demethylating DNA, but Kriaucionis and Heintz believe it may have a positive role in regulating gene expression as well. The reason that this nucleotide had not been seen before, the researchers say, is because of the methodologies used in most epigenetic experiments. Typically, scientists use a procedure called bisulfite sequencing to identify the sites of DNA methylation. But this test cannot distinguish between 5-hydroxymethylcytosine and 5-methylcytosine, a shortcoming that has kept the newly discovered nucleotide hidden for years, the researchers say. Its discovery may force investigators to revisit earlier work. The Human Epigenome Project, for example, is in the process of mapping all of the sites of methylation using bisulfite sequencing. "If it turns out in the future that (5-hydroxymethylcytosine and 5-methylcytosine) have different stable biological meanings, which we believe very likely, then epigenome mapping experiments will have to be repeated with the help of new tools that would distinguish the two," says Kriaucionis.

Providing further evidence for their case that 5-hydroxymethylcytosine is a serious epigenetic player, a second paper to be published in Science by an independent group at Harvard reveals the discovery of genes that produce enzymes that specifically convert 5-methylcytosine into 5-hydroxymethylcytosine. These enzymes may work in a way analogous to DNA methyltransferase, suggesting a dynamic system for regulating gene expression through 5-hydroxymethylcytosine. Kriaucionis and Heintz did not know of the other group's work, led by Anjana Rao, until earlier this month. "You look at our result, and the beautiful studies of the enzymology by Dr. Rao's group, and realize that you are at the tip of an iceberg of interesting biology and experimentation," says Heintz, a neuroscientist whose research has not focused on epigenetics in the past. "This finding of an enzyme that can convert 5-methylcytosine to 5-hydroxymethylcytosine establishes this new epigenetic mark as a central player in the field."

Kriaucionis is now mapping the sites where 5-hydroxymethylcytosine is present in the genome, and the researchers plan to genetically modify mice to under- or overexpress the newfound nucleotide in specific cell types in order to study its effects. "This is a major discovery in the field, and it is certain to be tied to neural function in a way that we can decipher," Heintz says.

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Journal reference:

1. Skirmantas Kriaucionis and Nathaniel Heintz. The Nuclear DNA Base 5-Hydroxymethylcytosine Is Present in Purkinje Neurons and the Brain. Science, 2009; DOI: 10.1126/science.1169786

Adapted from materials provided by Rockefeller University, via EurekAlert!, a service of AAAS.

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Human genome further unravelled

Human genome further unravelled

  8:05:00 PM    Science Lover

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A close-up view of the human genome has revealed its innermost workings to be far more complex than first thought.

The study, which was carried out on just 1% of our DNA code, challenges the view that genes are the main players in driving our biochemistry.

Instead, it suggests genes, so called junk DNA and other elements, together weave an intricate control network.

The work, published in the journals Nature and Genome Research, is to be scaled up to the rest of the genome.

Views transformed

The Encyclopaedia of DNA Elements (Encode) study was a collaborative effort between 80 organisations from around the world.

It has been described as the next step on from the Human Genome Project, which provided the sequence for all of the DNA that makes up the human species' biochemical "book of life".

We are now seeing the majority of the rest of the genome is active to some extent Tim Hubbard, Sanger Institute

Ewan Birney, from the European Molecular Biology Laboratory's European Bioinformatics Institute, led Encode's analysis effort. He told the BBC: "The Human Genome Project gave us the letters of the genome, but not a great deal of understanding. The Encode project tries to understand the genome."

The researchers focussed on 1% of the human genome sequence, carrying out 80 different types of experiments that generated more than 600 million data points.

The surprising results, explained Tim Hubbard from the Wellcome Trust Sanger Institute, "transform our view of the genome fabric".

THE DNA MOLECULE The double-stranded DNA molecule - wound in a helix - is held together by four chemical components called bases Adenine (A) bonds with thymine (T); cytosine(C) bonds with guanine (G) Groupings of these "letters" form the "code of life"; a code that is very nearly universal to all Earth's organisms Written in the DNA are genes which cells use as starting templates to make proteins; these sophisticated molecules build and maintain our bodies

Previously, genome activity was thought of in terms of the 22,000 genes that make proteins - the functional building blocks in our cells - along with patches of DNA that control, or regulate, the genes.

The other 97% or so of the genome was said to be made up of "junk" DNA - so called because it had no known biological function.

However, junk DNA may soon need a new moniker.

Dr Hubbard said: "We are now seeing the majority of the rest of the genome is active to some extent."

He explained that the study had found junk DNA was being transcribed, or copied, into RNA - an active molecule that relays information from DNA to the cellular machinery.

He added: "This is a remarkable finding, since most prior research suggested only a fraction of the genome was transcribed."

'Complex picture'

Dr Birney added that many of the RNA molecules were copying overlapping sequences of DNA.

He said: "The genome looks like it is far more of a network of RNA transcripts that are all collaborating together. Some go off and make proteins; [and] quite a few, although we know they are there, we really do not have a good understanding of what they do.

"This leads to a much more complex picture."

The researchers now hope to scale up their efforts to look at the other 99% of the genome.

By finding out more about its workings, scientists hope to have a better understanding of the mechanics of certain diseases.

Dr Birney said that in the future, they would hope to combine their findings with some of the larger studies that are currently investigating genes known to be associated with particular conditions.

He added: "As we understand these things better, we get better insight into disease, and when we get better insight into disease, we get better insight into diagnosis and the chances to create new drugs."..

SOURCE : BBC NEWS

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