The turn of the corkscrew : Structural analysis uncovers mechanisms of gene expression

The turn of the corkscrew : Structural analysis uncovers mechanisms of gene expression

  9:36:00 PM    Science Lover

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The diverse functions of living cells are all based on the information encoded in the structure of the hereditary material DNA. Gene expression must therefore be tightly controlled, and this task is accomplished by the binding of regulatory proteins to, and their removal from, specific DNA sequences. One class of large molecular machines known as Swi2/Snf2 remodelers plays a central role in modulating these processes. However, until now, it was not clear how Swi2/Snf2 remodelers actually work. A team led by Professor Karl-Peter Hopfner at the Gene Center at Ludwig-Maximilians-Universität (LMU) in Munich has clarified the structure and function of the remodeler Mot1 (Modifier of Transcription 1), which binds directly to DNA. It turns out that Mot1 acts like a molecular corkscrew that migrates along the DNA, following its helical contour. During its progress, Mot1 displaces a crucial transcription factor called TBP (for “TATA Box Binding Protein”) from the DNA. Removal of TBP from a TATA box represses transcription of the adjacent gene, and the protein encoded by that gene is no longer synthesized. At the same time, TBP is stabilized and its binding specificity is changed, which facilitates the expression of genes that lack TATA boxes and code for other proteins. (Nature 6 July 2011)

The DNA in the cells of higher organisms is tightly wrapped around protein complexes called nucleosomes. This type of structural organization not only makes it possible to package the long DNA molecules in a highly compact form, it also provides the basis for the controlled expression of genetic information. Densely packed sections of the molecule are effectively in a repressed state, and genes located in these DNA segments cannot be transcribed. Activation of repressed genes depends on the intervention of complex molecular machines, so-called Swi2/Snf2 remodelers, which reorganize condensed stretches of DNA so as to make them accessible for transcription. The precise mode of action of remodelers has so far been unclear, mainly because most of them are made up of several components and the active complexes are difficult to study. This is why Hopfner chose to study Mot1, which is a comparatively simple representative of the family that functions as an Swi2/Snf2 remodeler on its own, and can serve as a guide to understanding the more complicated members of the class. Mot1 is known to participate in the control of gene expression, but how exactly it does so is not well understood.

The first stage of the process that leads to the synthesis of a given protein is the transcription of the specific segment of DNA that codes for it into molecules of messenger RNA. This initial step requires the action of so-called transcription factors. One of the most important of these is TBP, which binds preferentially to DNA sequences called TATA boxes that are located near the beginnings of many genes. Binding of TBP introduces a kink into the DNA, and this landmark serves as a platform for the binding of further proteins, ultimately leading to the assembly of the complex necessary for the initiation of transcription. Mot1 regulates transcription by actively removing TBP from the DNA, using ATP as a source of energy. “How Mot1 dissociates the TBP-DNA complex was completely unclear up to now,” says Hopfner. With the aid of so-called hybrid methods - in which data obtained from high-resolution X-ray diffraction analysis of the crystallized protein complex with images of the same molecular complex taken with the electron microscope were combined - Hopfner’s team was able to define the three-dimensional structure of the Mot1-TBP complex for the first time. This revealed how Mot1 recognizes the surface of the DNA-bound TBP. “Once Mot1 has recognized TBP, it binds to the adjacent DNA and begins to migrate along the DNA strand, using the energy released by the hydrolysis of ATP to power its movement. This helical movement, which is reminiscent of the insertion of a corkscrew, causes TBP to detach from the DNA,” explains Dr Petra Wollmann, who is first author on the new study. The researchers were surprised to find that Mot1 contains a strikingly extended loop. After TBP has dissociated from the DNA, this loop masks TBP’s DNA binding site and prevents the protein from reoccupying it.



Previous studies had reported what appeared to be paradoxical observations, which indicated that Mot1 inhibits transcription of TATA box-containing genes while facilitating the expression of genes that lack canonical TATA boxes. “Our results suggest that Mot1 also stabilizes the DNA-free conformation of TBP, increasing the probability that it can reach, bind to and activate genes that lack TATA boxes,” explains Hopfner. In other words, Mot1 is also a redistribution factor, which enables TBP to bind to different sequences and thus controls its association with other cellular components. This combination of detachment and redistribution functions may be a common feature of remodeling complexes, and would help to explain how they mediate the large-scale redistribution of DNA-binding regulatory proteins. (göd/PH)

The project was carried out under the auspices of two Clusters of Excellence - the Center for Integrated Protein Science Munich (CiPSM) and the Munich Centre for Advanced Photonics (MAP). The work was also supported by the German Research Foundation (DFG) as part of the Collaborate Research Centers (SFB) 646 and TR5, and by the LMUexcellent Investment Fund.

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Non-coding RNA has role in inherited neurological disorder -- and maybe other brain diseases too

Non-coding RNA has role in inherited neurological disorder -- and maybe other brain diseases too

  1:26:00 AM    Science Lover

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A team of scientists, led by researchers at the University of California, San Diego School of Medicine, have uncovered a novel mechanism regulating gene expression and transcription linked to Spinocerebellar ataxia 7, an inherited neurological disorder. The discovery promises to have broad ramifications, suggesting that abundant non-coding transcripts of ribonucleic acid (RNA) may be key players in neurological development and function, and could be powerful targets for future clinical therapies.

Researchers have discovered that expression
of the ataxin‑7 gene - the cause of the
neurological disorder spinocerebellar
ataxia type 7 - has two regulators: a highly
conserved, multi‑tasking protein called
CTCF and, surprisingly, an adjacent promoter
containing non‑coding RNA.
Credit: Illustration courtesy of Christina
Takamatsu‑Butler, UC San Diego.

The research, headed by Albert La Spada, MD, PhD, chief of the division of genetics in the UCSD department of pediatrics, and professor of cellular and molecular medicine, neurosciences and biological sciences, is published in the June 22 issue of the journal Neuron.

"Our paper highlights a number of important emerging themes in our understanding of gene regulation in the brain," said La Spada, who is also associate director of the UCSD Institute for Genomic Medicine.

"With the advent of new technologies, science has learned that the vast majority of our transcripts are non-coding," said La Spada. "The challenge going forward is to determine what they do do, and if they have specific functions. It now seems increasingly likely that a multitude of these non-coding RNAs help finely tune transcription regulation in the brain, and perturbation of their work is linked to disease. If we can figure out exactly how, we should be able to gain new insights into how the brain is so precisely regulated – knowledge that may help us better understand how the brain works."



Spinocerebellar ataxia 7 is one of several types of spinocerebellar ataxia (SCA), genetic degenerative disorders characterized by atrophy in the cerebellum of the brain, progressive loss of physical coordination – and in the case of type 7 – retinal degeneration that can result in blindness. There is currently no known cure.

Many SCAs are classified as polyglutamine diseases, caused when a protein associated with the disease contains too many repeats of the amino acid glutamine. Polyglutamine diseases are also known as "CAG Triplet Repeat Disorders" because CAG is the sequence of nucleic acids that codes for glutamine.

La Spada and colleagues have long studied SCA. In 2001, they were the first to demonstrate that SCA7 retinal degeneration was the result of transcription dysregulation of ataxin-7, the protein associated with SCA7. Following up, they decided to learn how the gene that expresses ataxin-7 is itself regulated.

The researchers found not one, but two, regulators. The first is called CTCF, a highly conserved protein that regulates a variety of transcriptional processes, most notable establishing insulator domains and controlling genomic imprinting. But they also discovered an adjacent, alternative promoter dubbed intron 2 promoter (P2A) and a transcribed antisense, non-coding RNA, which they labeled SpinoCerebellarAtaxia-AntisenseNoncodingTranscript1 or SCAANT1.

Antisense RNA is single-stranded ribonucleic acid whose primary function appears to be as an inhibitor or suppressor of a gene, though sometimes it can promote gene expression instead. Most antisense RNAs are non-coding, meaning that their sequences do not provide information for making proteins. Even though non-coding RNAs do not provide instructions for the production of vital proteins, they comprise the bulk of the human genome. A major challenge for biomedical research in the 21st century is to figure what they do, and how they do it.

In their Neuron paper, La Spada and colleagues highlight one function, at least for SCAANT1. When they investigated how CTCF regulated ataxin-7 gene expression in transgenic mice, they discovered that CTCF promotes the production of SCAANT1 which in turn represses the newly discovered ataxin-7 sense promoter P2A. In mice lacking SCAANT1, sense promoter P2A is de-repressed, allowing a mutant ataxin-7 gene to be expressed, resulting in mice with a version of SCA7. The scientists found a similar lack of antisense SCAANT1 in the fibroblasts and white blood cells taken from human patients with SCA7, implicating deregulation of this pathway in the disease process.

As many inherited neurological disorders are now known to exhibit such overlapping "bidirectional" transcription, the findings in SCA7 could shed light on similar abnormalities with non-coding RNA function in a number of brain diseases.

Provided by University of California - San Diego

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Genetic 'Parts' List Now Available for Hypothalamus

Genetic 'Parts' List Now Available for Hypothalamus

  8:41:00 PM    Science Lover

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A Johns Hopkins and Japanese research team has generated the first comprehensive genetic "parts" list of a mouse hypothalamus, an enigmatic region of the brain -- roughly cherry-sized, in humans -- that controls hunger, thirst, fatigue, body temperature, wake-sleep cycles and links the central nervous system to control of hormone levels.

A. A diagram of the developing mouse brain at 12 
days gestation. The hypothalamus is outlined in 
red, while cells that express the Shh gene, which 
was used as a landmark in this study, are shown in 
brown. B. Various different genes that are turned on 
in different parts of the hypothalamus are shown in 
purple, while Shh is shown in brown. (Credit: Image 
courtesy of Johns Hopkins Medical Institutions)

Flaws in hypothalamus development may underlie both inborn and acquired metabolic balance problems that can lead to obesity, diabetes, mood disorders and high blood pressure, according to a report on the study published May 2 in the advance online publication of Nature Neuroscience.

"Knowing how cells develop in this part of the brain will help us understand how they regulate behavior, mood and metabolism," says Seth Blackshaw, Ph.D., an assistant professor in the Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine.

The hypothalamus is one of the most diverse and complex parts of the brain, and having an index of the genes involved in producing its many cell types is a toolbox that researchers can use to manipulate the activity of brain cells by turning them on and off, or tracing their connections. This may ultimately lead to better diagnostic and treatment options for a variety of disorders.

"The study of hypothalamic development, particularly of cell specification, will help us to understand how hypothalamic neurons function to regulate behavior and physiology," says Blackshaw. "Because of when and where we saw certain genes turn on, we now have identified a set of candidate players that guide the assembly of the different parts of the hypothalamus and that specify the many individual cell types within it."

The hypothalamus is composed of at least dozens of types of neurons -- and more likely hundreds -- each of which corresponds to a gene that has remained unidentified until now. Its cellular arrangement is more akin to a bowl of spaghetti than a neatly organized club sandwich, according to Blackshaw. The catalog of molecular markers identified here helps unravel this complexity.

The team's first challenge was to dissect away, at the very start of neural development, the part of the mouse brain which develops into the hypothalamus, and then cut tiny slices of this region for use in microarray analysis, a technology that reveals multiple gene activity. By analyzing all the roughly 20,000 genes in the mouse genome, the team identified 1200 as strongly activated in developing hypothalamus and characterized the cells within the hypothalamus in which they were activated. The team then characterized the expression of the most interesting 350 genes in detail using another gene called Shh, for sonic hedgehog, as a landmark to identify the precise region of the hypothalamus in which these genes were turned on. This involved processing close to 20,000 tissue sections -- painstakingly sliced at one-fiftieth of a millimeter thickness and then individually examined.

"We were able to use this data to find genes whose expression matched every individual hypothalamic nucleus and essentially assemble a jigsaw puzzle of gene expression patterns that completely covered the developing hypothalamus," Blackshaw says. "Now that we have a complete set of molecular landmarks, along with an extensive molecular parts list, we can begin to learn how all these parts fit together to create this essential and highly complex brain region."

Authors of the paper, in addition to Blackshaw, are Daniel A. Lee, Ana Miranda-Angulo, Yangqin Yang, Aya C. Yoshida, Hong Wang, Hiromi Mashiko, Lizhi Jiang, Marina Avetisyan, Lixin Qi, and Jiang Qian, all of Johns Hopkins; Ayane Kataoka and Tomomi Shimogori of RIKEN-BSI, 2-1 Hirosawa, Wako-shi, Saitama, Japan.

This research was supported by March of Dimes, the Klingenstein Fund, the W.M. Keck Foundation, and the Japan Society for Promotion of Science.

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What Makes You Unique? Not Genes So Much as Surrounding Sequences, Study Finds

What Makes You Unique? Not Genes So Much as Surrounding Sequences, Study Finds

  10:27:00 AM    Science Lover

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The key to human individuality may lie not in our genes, but in the sequences that surround and control them, according to new research by scientists at the Stanford University School of Medicine and Yale University. The interaction of those sequences with a class of key proteins, called transcription factors, can vary significantly between two people and are likely to affect our appearance, our development and even our predisposition to certain diseases, the study found.

Researchers have found that the unique, specific changes among individuals in the sequence of DNA affect the ability of "control proteins" called transcription factors to bind to the regions that control gene expression. (Credit: iStockphoto/Andrey Prokhorov)

The discovery suggests that researchers focusing exclusively on genes to learn what makes people different from one another have been looking in the wrong place.

"We are rapidly entering a time when nearly anyone can have his or her genome sequenced," said Michael Snyder, PhD, professor and chair of genetics at Stanford. "However, the bulk of the differences among individuals are not found in the genes themselves, but in regions we know relatively little about. Now we see that these differences profoundly impact protein binding and gene expression."

Snyder is the senior author of two papers -- one in Science Express and one in Nature -- exploring these protein-binding differences in humans, chimpanzees and yeast. Snyder, the Stanford W. Ascherman, MD, FACS, Professor in Genetics, came to Stanford in July 2009 from Yale, where much of the work was conducted.

Genes, which carry the specific instructions necessary to make proteins do the work of the cell, vary by only about 0.025 percent across all humans. Scientists have spent decades trying to understand how these tiny differences affect who we are and what we become. In contrast, non-coding regions of the genome, which account for approximately 98 percent of our DNA, vary in their sequence by about 1 to 4 percent. But until recently, scientists had little, if any, idea what these regions do and how they contribute to the "special sauce" that makes me, me, and you, you.

Now Snyder and his colleagues have found that the unique, specific changes among individuals in the sequence of DNA affect the ability of "control proteins" called transcription factors to bind to the regions that control gene expression. As a result, the subsequent expression of nearby genes can vary significantly.

"People have done a lot of work over the years to characterize differences in gene expression among individuals," said Snyder. "We're the first to look at differences in transcription-factor binding from person to person." What's more, by selectively breeding, or crossing, yeast strains, Snyder and his colleagues found that many, but not all, of these differences in binding and expression levels are heritable.

In the Science Express paper, which will be published online March 18, Snyder and his colleagues compared the binding patterns of two transcription factors in 10 people and one chimpanzee. They identified more than 15,000 binding sites across the genome for the transcription factor called NF-kB and more than 19,000 sites for another factor called RNA PolII. They then looked to see if every site was bound equally strongly by the proteins, or if there were variations among individuals.

They found that about 25 percent of the PolII sites and 7.5 percent of the NF-kB sites exhibited significant binding differences among individuals -- in some cases greater than two orders of magnitude from one person to another. (For comparison, the binding differences between the humans and the chimpanzee were about 32 percent.) Many of these binding differences could be traced to differences in sequences or structure in the protein binding sites, and several were directly correlated to changes in gene expression levels.

"These binding regions, or chunks, vary among individuals," said Snyder, "and they have a profound impact on gene expression." In particular, the researchers found that several of the variable binding regions were near genes involved in such diseases as type-1 diabetes, lupus, leukemia and schizophrenia.

The researchers confirmed and extended their findings in the Nature paper, which will be published online March 17. In this study, they used yeast to determine that many of the binding differences and variations in gene expression levels in individuals are passed from parent to progeny, and they identify several control proteins that vary -- a study that would have been impossible to perform in humans.

"We conducted the two studies in parallel," said Snyder, "and found the same thing. Many of the binding sites differed. When we mapped the areas of difference, we found that they were associated with key regulators of variation in the population. Together these two studies tell us a lot about the so-called regulatory code that controls variation among individuals."

The research in the Science Express study was supported by the National Institutes of Health, the European Molecular Biology Laboratory and the Howard Hughes Medical Institute's Medical Fellows Program. The research in the Nature study was supported by the National Institutes of Health. In addition to Snyder, other Stanford researchers involved in the two studies include postdoctoral scholars Fabian Grubert, PhD; Minyi Shi, PhD; and Manoj Hariharan, PhD; and graduate student Konrad Karczewski.

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First Transgenic Prairie Voles May Help Unlock Secrets of Pair Bonding

First Transgenic Prairie Voles May Help Unlock Secrets of Pair Bonding

  1:00:00 AM    Science Lover

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Researchers at the Yerkes National Primate Research Center, Emory University, have successfully generated the first transgenic prairie voles, an important step toward unlocking the genetic secrets of pair bonding. The future application of this technology will enable scientists to perform a host of genetic manipulations that will help identify the brain mechanisms of social bonding and other complex social behaviors.

Researchers have successfully generated the first transgenic prairie voles. (Credit: Zoe Donaldson, Yerkes National Primate Research Center)

This advancement may also have important implications for understanding and treating psychiatric disorders associated with impairments in social behavior.

The study is available in the December issue of Biology of Reproduction.

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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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