Scientists show first 3-D image of antibody gene

Using a multidisciplinary mix of geometry, biological research and techniques developed to solve problems on supercomputers, scientists at the University of California, San Diego have shown for the first time how a genome is organized in three-dimensional space.

The 3-D structure of the immunoglobulin locus in B cells is shown, with the relative positions of the different portions of the immunoglobulin genes. Grey objects indicate constant regions. Blue objects indicate proximal variable regions. Green objects indicate distal variable regions. Red line indicates the linker connecting the proximal variable and joining regions.

Researchers led by Cornelis Murre, a professor of biology at UC San Diego, and Steve Cutchin, senior scientist for visualization services at the San Diego Supercomputer Center (SDSC), used the gene encoding the immunoglobulin heavy chain locus — responsible for generating diverse kinds of antibodies — to demonstrate the structure of the genome.

The observations, the researchers say, permit an insight into the structure of the human genome, which until now has remained elusive.

Their results, “The 3-D Structure of the Immunoglobulin Heavy Chain Locus: Implications for Long-Range Genomic Interactions,” are published in the April 18 issue of the journal Cell.

Because the genome is the most essential part of the cell for storing and accessing genetic information, the complete DNA sequence of a wide variety of genomes has been revealed in studies performed in a large number of laboratories — “a tremendous success that has provided insight into mechanisms that underpin the development of a wide variety of diseases,” the authors say.

However, Murre said, “it has remained unclear as to how the genome is organized in three-dimensional space. This is an important issue since the regulation of gene expression is controlled by interactions of genomic elements that are separated by large genomic distances. Thus, our team wanted to determine how the genome is structured within the nucleus.”

The experiments described in the Cell paper, he said, provide a first glimpse into this question. “As a model system, we used the gene encoding for the immunoglobulin heavy chain locus, because it is responsible for generating the wide diversity of antibodies.”

Having measured the distances that separate the various parts of the gene, Murre said, the researchers, in collaboration with Cutchin at the SDSC, then used geometry to resolve the first structure of a genetic locus.

His work, said Cutchin, involved computational geometry, scientific visualization, computational methods and numerical methods.

“The resulting structure shows that the antibody gene is organized into ‘flower-like’ structures that are connected by linkers,” said Murre. “These flowers contain the various parts that ultimately generate the wide variety of antibodies. This is the first time that geometry has been used to determine the structure of a genetic locus. Ultimately, the same approach should be used to elucidate the structure of the entire human genome.”

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Discovery of the First Sex Chromosome Gene Tied to Male Infertility

A team of scientists led by University of Pennsylvania veterinary researchers have identified a gene, TEX11, located on the X chromosome, which when disrupted in mice renders the males sterile and reduces female fecundity. This is the first study of the genetic causes of infertility that links a particular sex chromosome meiosis-specific gene to sterility.

As with mice, the TEX11 gene is also located on the human X chromosome. Given that disruption of TEX11 causes azoospermia, or non-measurable sperm levels in mice, mutations in the human TEX11 gene may be a genetic cause of infertility in men. Because men have only one X chromosome that they inherit from their mother and thus only one copy of the TEX11 gene, any mutation could theoretically lead to sterility. Like other X-linked disorders such as color blindness and muscular dystrophy, genetic mutation causing a son’s infertility could be passed from his mother.

Researchers hypothesize that a screening of the TEX11 gene may provide a pre-birth diagnosis for infertility in men.

The study, published in the March issue of Genes & Development, also reports the first meiosis-specific factor ever found on the X chromosome. Meiosis is the process of cell division that produces gametes in both sexes. During meiosis, homologous chromosomes undergo pairing, synapsis, recombination and faithful segregation. Meiosis allows the exchange of genetic material between paternal and maternal genomes to produce genetically diverse gametes (sperm or eggs). Therefore, defects in meiosis are a leading cause of both infertility and birth defects.

An estimated 15 percent of couples are affected by infertility worldwide, yet the genetic causes of male infertility remain largely unknown. For decades, conventional wisdom stated that the X chromosome had little to do with meiosis or infertility because the X chromosome is silenced during male meiosis. This thinking led to fertility studies that focused on the Y chromosome and autosomes.

In fact, Jeremy Wang, assistant professor in the Department of Animal Biology at the University of Pennsylvania’s School of Veterinary Medicine, and his team revealed in an earlier study of mouse male germ cells that nearly one third of the germ cell-specific genes they identified are located on the X-chromosome.

Wang and his team found that sex chromosomes did play a role in meiosis. Although these X-linked, germ cell-specific genes undergo inactivation during later stages of male meiosis, they play a role in the early stages. Specifically, researchers found that TEX11 forms discrete foci on meiotic chromosomes and appears to be a novel constituent of the meiotic recombination machinery. The team genetically engineered male mice such that they lacked TEX11 function and found that this caused chromosomal asynapsis during the process of gamete formation.

This means that homologous chromosomes failed to pair together during meiosis and chromosomes formed fewer crossovers, i.e. sites where they recombine, during the initial stages of meiosis. These failures led to elimination of spermatocytes at later stages in the genetic recombination process and, ultimately, male infertility.

Researchers hypothesize that because TEX11 interacts with SYCP2, an integral component of the protein complex that mediates synapsis during meiosis, TEX11 promotes both synapsis and genetic recombination and may provide a physical link between these two meiotic processes.

The study was performed by Wang and Fang Yang in the Department of Animal Biology in Penn’s School of Veterinary Medicine; Katarina Gell and Christer Höög of the Department of Cell and Molecular Biology at the Karolinska Institutet; Godfried W. van der Heijden and David C. Page of the Howard Hughes Medical Institute, Whitehead Institute and Department of Biology of the Massachusetts Institute of Technology; Sigrid Eckardt, N. Adrian Leu and K. John McLaughlin of the Center for Animal Transgenesis and Germ Cell Research at Penn Vet’s New Bolton Center; Ricardo Benavente of the Department of Cell and Developmental Biology at the University of Würzburg; and Chengtao Her of the School of Molecular Biosciences and Center for Reproductive Biology at Washington State University.
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Gene's 'selective signature' helps scientists identify instances of natural selection in microbial evolution

Microbes, the oldest and most numerous creatures on Earth, have a rich genomic history that offers clues to changes in the environment that have occurred over hundreds of millions of years.

While scientists are becoming increasingly aware of the many important environmental roles played by microbes living today--they process the food in our intestines, they keep carbon moving through the ocean food web, they can be harnessed to process sewage and build specific proteins--they still know little about these tiny critters, particularly marine microbes, which generally are classified into species based on their ecological niche. For instance, two species of marine microbe might look very similar physically, but one may have adapted to life in a particularly dark part of the ocean, while its sister species may have adapted to feeding off a nutrient that is rare in most parts of the ocean, but exists in abundance in one small area.

Scientists at MIT who are trying to understand existing microbes by studying their genetic history recently created a new approach to the study of microbial genomes that may hasten our collective understanding of microbial evolution.

The researchers have reversed the usual order of inquiry, which is to study an organism, then try to identify which proteins and genes are involved in a particular function. Instead, they have come up with a simple mathematical formula that makes it possible to analyze a gene family (a single type of gene or protein that exists in many creatures) simultaneously in a group of ecologically distinct species.

This means that we can begin to identify occurrences of natural selection in an organism's evolution simply by looking at its genome and comparing it with many others at once. This would allow them to take advantage of the nearly 2,500 microbes whose genomes have already been sequenced.

The new method determines the "selective signature" of a gene, that is, the pattern of fast or slow evolution of that gene across a group of species, and uses that signature to infer gene function or to map changes to shifts in an organism's environment.

"By comparing across species, we looked for changes in genes that reflect natural selection and then asked, 'How does this gene relate to the ecology of the species it occurs in?'" said Eric Alm, the Doherty Assistant Professor of Ocean Utilization in the Departments of Civil and Environmental Engineering and Biological Engineering. Natural selection occurs when a random genetic mutation helps an organism survive and becomes fixed in the population. "The selective signature method also allows us to focus on a single species and better understand the selective pressures on it," said Alm.

"Our hope is that other researchers will take this tool and apply it to sets of related species with fully sequenced genomes to understand the genetic basis of that ecological divergence," said graduate student B. Jesse Shapiro, who coauthored with Alm a paper published in the February issue of PLoS Genetics.

Their work also suggests that evolution occurs on functional modules--genes that may not sit together on the genome, but that encode proteins that perform similar functions.

"When we see similar results across all the genes in a pathway, it suggests the genomic landscape may be organized into functional modules even at the level of natural selection," said Alm. "If that's true, it may be easier than expected to understand the complex evolutionary pressures on a cell."

For example, in Idiomarina loihiensis, a marine bacterium that has adapted to life near sulfurous hydrothermal vents in the ocean floor, the genes involved in metabolizing sugar and the amino acid phenylalanine underwent significant changes (over hundreds of millions of years) that may help the bacterium obtain carbon from amino acids rather than from sugars, a necessity for life in that ecological niche. In one of I. loihiensis' sister species, Colwellia psychrerythraea, some of those same genes have been lost altogether, an indication that sugar metabolism is no longer important for Colwellia.

Shapiro and Alm focused on 744 protein families among 30 species of gamma-proteobacteria that shared a common ancestor roughly one to two billion years ago. These bacteria include the laboratory model organism E. coli, as well as intracellular parasites of aphids, pathogens like the bacteria that cause cholera, and soil and plant bacteria. They mapped the evolutionary distance of each species from the ancestor and incorporated information about the gene family (for instance, important proteins evolve more slowly than less-vital ones) and the normal rate of evolution in a particular species' genome in order to determine a gene's selective signature.

"These are experiments we could never perform in a lab," said Alm. "But Mother Nature has put genes into an environment and run an evolutionary experiment over billions of years. What we're doing is mining that data to see if genes that perform a similar function, say motility, evolve at the same rate in different species. To the extent that they differ, it helps us to understand how change in core genes drives functional divergence between species across the tree of life."

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Breast cancer gene carriers’ risk ‘amplified’ by additional genes

Many women with a faulty breast cancer gene could be at greater risk of the disease due to extra ‘risk amplifying’ genes, according to research published in this week in the American Journal of Human Genetics.

Cancer Research UK scientists, including lead author Professor Doug Easton at the University of Cambridge, have found that common versions of two genes – FGFR2 and TNRC9 – known to increase breast cancer risk in the general population - also increase the risk in women carrying damaged versions of the BRCA2 gene.

Around one in eighteen women will develop breast cancer by the age of 65. On average, half of women carrying a faulty BRCA2 gene will develop the disease by the age of 70.

This study found that particular combinations of the FGFR2 and TNRC9 genes modify the breast cancer risk in BRCA2 mutation carriers.

Around twenty percent of the BRCA2 mutation carriers have the lowest risk combination of the FGFR2 and TNRC9 genes. The researchers found that their risk is lowered so four in every 10 women in this category are expected to develop breast cancer.

But one per cent of BRCA2 mutation carriers have the highest risk combination of FGFR2 and TNRC9 genes. Seven in every 10 women in this category are predicted to develop the disease.

These findings are the first step in a series of studies hunting for breast cancer susceptibility genes, which aims to better monitor and treat women with a family history of the disease.

Professor Easton, director of Cancer Research UK’s Genetic Epidemiology Unit at the University of Cambridge, said: “This is the first time we have found evidence that common changes in other genes can amplify the risk of breast cancer in women known to have faulty BRCA genes.

“This is the first step in finding a set of genes that modify the risk in BRCA carriers, and may influe nce how we monitor women with a family history of the disease.”

The study brings together results from international research groups looking at a total of more than 10,000 women carrying a BRCA1 or BRCA2 mutation. Dr Lesley Walker, Cancer Research UK’s director of cancer information, said: “It’s important to remember that the prevalence of this combination of gene faults is rare in the general population. But advances like this will add to our ability to identify those most at risk for clinical monitoring, detecting the disease earlier in those who develop it.”
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A Protein that Triggers Aggressive Breast Cancer

SATB1 is a nuclear protein well known for its crucial role in regulating gene expression during the differentiation and activation of T cells, making it a key player in the immune system. But SATB1 has now revealed a darker side: it is an essential contributing factor in the most aggressive forms of breast cancer.

SATB1 forms a three-dimensional cage-like structure within the cell nucleus (left) that binds to DNA specific sites within genes, reorganizes chromatin, and recruits enzymes that promote the expression or suppression of genes (right).

Breast cancer cells need SATB1 to become metastatic; metastasis — the stage when cells break away from the original tumor and spread to other parts of the body — is the final step of solid tumor progression and is the most common cause of death in cancer patients.

"In breast tumors, SATB1 reprograms the genome to change the expression of hundreds of genes, promoting tumor growth and metastasis," says Terumi Kohwi-Shigematsu, a scientist in the Life Sciences Division of the Department of Energy's Lawrence Berkeley National Laboratory who, with her colleagues, discovered SATB1 and has since investigated its many functions. She says, "SATB1's role in breast cancer is a new paradigm for the way tumors progress."

Kohwi-Shigematsu, working with Berkeley Lab's Hye-Jung Han and Yoshinori Kohwi, and with Jose Russo of the Fox Chase Cancer Center in Philadelphia, found that when SATB1 is detected in a breast tumor, the cancer is highly likely to progress or recur.

Moreover, by introducing SATB1 into otherwise nonmetastatic breast cancer cells, invasive tumors can be induced in mice; conversely, removing SATB1 from metastatic cells not only abolishes metastasis and tumor growth in mice but also returns cells to their normal appearance in vitro. The researchers have published these and other findings in the March 13, 2008 issue of Nature.

How SATB1 works

Kohwi-Shigematsu and Kohwi originally identified a class of DNA sequences they called base-unpairing regions (BURs), a finding that led Kohwi-Shigematsu's group to the discovery of "special AT-rich sequence binding protein 1" (SATB1). SATB1 binds to BURs in double-stranded DNA by recognizing the BURs' distinctive phosphate-backbone structure. BURs contain unusual sequence contexts that readily unzip to expose DNA's individual strands.

As a nuclear architectural protein, SATB1 forms what Kohwi-Shigematsu calls a "3‑D chickenwire network" inside the nucleus of the cell. SATB1 anchors chromosomes to its cage-like structure by tethering the BURs in the target genes, thus serving as a kind of "glue" for these genes. SATB1 folds and remodels the chromatin — the intertwined DNA and proteins that form chromosomes — into new shapes, bringing even distant parts of the genome together for coordinated control of gene expression and regulation.

SATB1 also globally regulates histone status in the chromatin by recruiting histone-modifying enzymes to the target-gene loci. Histones are the proteins around which DNA in chromatin is wound like thread on a spool; histone status renders DNA sequences accessible or inaccessible for transcription.

Early on, SATB1's ability to regulate gene expression was identified as critical to T-cell development. Although Kohwi-Shigematsu and her colleagues have found several other cell types that use SATB1 to reshape chromatin and regulate gene expression in a similar way, SATB1 is not expressed in all cells. SATB1 seems particularly important in cells which must change their function — as do many progenitor cells, including the thymocytes that turn into T cells. And as cancerous cells must do to turn into metastatic cells.

"Hye-Jung Han of our group started by looking at two dozen breast-cell lines, including normal human epithelial cells" — epithelial cells are the kind that form the linings of hollow glands in the breast — "and both nonmetastatic and metastatic breast cancer cells," Kohwi-Shigematsu says. "Only the metastatic cells expressed SATB1, with the most aggressive breast cancer cells showing the highest levels of the protein."

The researchers examined over 2,000 human primary breast cancer tissue samples for which clinical follow-up studies were available. The highest levels of SATB1 were in samples from patients whose survival times had been shortest; patients whose tumor samples had no SATB1 expression generally had longer survival times.

The analysis showed that a high level of SATB1 expression by itself is an excellent indicator of poor prognosis — independent of whether breast cancer cells have already metastasized to the lymph nodes at the time of diagnosis.

SATB1 takes command

The reason why SATB1 is a good prognostic marker is because SATB1 drives breast cancer cells to become invasive, as revealed by both in vitro and in vivo studies.

The researchers performed in vitro studies of highly metastatic cell lines, reducing SATB1 expression through the use of shRNAs, "short-hairpin-interfering" RNAs, that dramatically reduced the invasive capacity of these cells and also reduced their capacity for unattached growth — a necessity if metastasizing cancer cells are to travel through the blood and lymph vessels.

With increasing stages of breast cancer, initially nonmalignant cells making up the acini in the breast become increasingly disorganized and finally metastasize (top). Breast cells cultured in a three-dimensional matrix form acinar structures, but the expression of SATB1 in these cells causes increasing disorganization. When SATB1 expression is reduced in these metastatic cells, they return to normal appearance (bottom).

Other in vitro studies used normal breast epithelial cells, the kind that form the hollow oriented structures called acini, the milk-secreting glands of the breast. Normal cells form similar, well-organized acinar structures in vitro, whereas in highly metastatic epithelial cell lines these structures are disorganized and lack polarity. When SATB1 expression is reduced in the metastatic cell lines, they too form the kind of polarized, uniform acinar structures found in normal mammary epithelial cells.

These in vitro results were confirmed in vivo, in mice. Nine weeks after human aggressive breast cancer cells were injected into the tails of test mice, these cells developed into metastatic nodules (tumors) on the lungs. But when SATB1 expression was reduced or removed from the injected cancer cells, the mice developed fewer or even no nodules, depending on the remaining levels of SATB1. Once SATB1 is greatly reduced, these cells no longer form tumors when injected directly into breast fat pads.

In vivo studies also established that cancer cells which do not normally express SATB1, and do not normally metastasize, can become aggressive if they are modified to express SATB1. Once SATB1 is expressed, they form large tumors when injected into breast fat pads; they then invade the blood circulatory system and form metastatic tumors in lungs.

The tests of human breast cancer cell lines in mice allowed the researchers to establish that, for these cells, SATB1 is necessary and sufficient for tumor growth and metastatic activity.

"SATB1 is a key player in the metastasis of breast cancer cells, controlling expression of over a thousand genes," says Kohwi-Shigematsu. "It increases the expression of genes that promote tumor growth and reduces the expression of tumor suppressors. Among the regulated genes are numerous growth-factor genes and genes affecting cell adhesion, cell signaling, cell-cycle regulation, and other functions."

Among the important genes regulated by SATB1, the researcher identified many that are already known to play a role in aggressive breast cancers, including the epidermal growth factor gene ERBB2, otherwise known as HER2.

"What we have found is a new model of altered gene regulation during the progression of tumors, which depends on SATB1's reprogramming of the gene expression profile," Kohwi-Shigematsu says. "What results is a new and aggressive cancer phenotype that promotes both tumor growth and metastasis."

The discovery of SATB1's key part in aggressive breast cancer has profound implications for prognosis and for possible new treatments for cancer's most malignant forms. At the same time, the discovery opens a wide field of fundamental scientific inquiry, beginning with the most basic questions. What determines the particular sets of genes affected by SATB1 in specific tissues? What other factors may work together with SATB1?

"An important question is what turns on SATB1 during breast cancer progression," says Kohwi-Shigematsu. "That's just the beginning of the things we really want to know."

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On a 'roll': MIT researchers devise new cell-sorting system

Capitalizing on a cell's ability to roll along a surface, MIT researchers have developed a simple, inexpensive system to sort different kinds of cells--a process that could result in low-cost tools to test for diseases such as cancer, even in remote locations.

Rohit Karnik, an MIT assistant professor of mechanical engineering and lead author of a paper on the new finding appearing last week in the journal Nano Letters, said the cell-sorting method was minimally invasive and highly innovative.

"It's a new discovery," he said. "Nobody has ever done anything like this before."

The method relies on the way cells sometimes interact with a surface (such as the wall of a blood vessel) by rolling along it. In the new device, a surface is coated with lines of a material that interacts with the cells, making it seem sticky to specific types of cells. The sticky lines are oriented diagonally to the flow of cell-containing fluid passing over the surface, so as certain kinds of cells respond to the coating they are nudged to one side, allowing them to be separated out.

Cancer cells, for example, can be separated from normal cells by this method, which could ultimately lead to a simple device for cancer screening. Stem cells also exhibit the same kind of selective response, so such devices could eventually be used in research labs to concentrate these cells for further study.

Normally, it takes an array of lab equipment and several separate steps to achieve this kind of separation of cells. This can make such methods impractical for widespread screening of blood samples in the field, especially in remote areas. "Our system is tailor-made for analysis of blood," Karnik said. In addition, some kinds of cells, including stem cells, are very sensitive to external conditions, so this system could allow them to be concentrated with much less damage than with conventional multistage lab techniques.

"If you're out in the field and you want to diagnose something, you don't want to have to do several steps," Karnik said. With the new system, "you can sort cells in a very simple way, without processing."

Now that the basic principle has been harnessed in the lab, Karnik estimates it may take up to two years to develop into a standard device that could be used for laboratory research purposes. Because of the need for extensive testing, development of a device for clinical use could take between five and 10 years, he estimated.

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Policing cells demand ID to tell friend from foe, say University of Pennsylvania cell engineers

University of Pennsylvania scientists studying macrophages, the biological cells that spring from white blood cells to eat and destroy foreign or dying cells, have discovered how these �policemen� differentiate between friend and foe.

The paper appears as the cover article in the March 10 edition of the Journal of Cell Biology.

The knowledge suggests new ways science may be able to turn off rogue macrophages that are the root cause of the many inflammatory diseases ranging from atherosclerosis to arthritis and that provide the mechanism for tissue and organ rejection after transplant.

There is also evidence that some types of cancer cells over-express the molecular protein that macrophages recognize as friendly � like a fake ID � which allows the cancer to avoid being perceived as foreign. In addition, the molecules involved in the recognition mechanism appear somewhat variable from person to person, with possible links to success or failure in transplantation of stem cells.

Researchers studied these main �policing cells� in the body, macrophages, from the Greek for �eating cells,� and how they choose to attack and engulf foreign cells and particles but recognize and refuse to eat their own kind.

�Like a police dog, a macrophage responds to a signal that means �let go,� Dennis Discher, professor of chemical and biomolecular engineering at Penn, said. �It�s what makes your body more of a 'dog eat cat' kind of world rather than a 'dog eat dog' world.�

Discher and his team observed macrophages in culture using �friendly� human blood cells and �foreign� sheep blood cells as well as plastic particles. In all cases, the macrophage binds to a target, often identified by a cloak of common IgG antibodies (the community watch), and �frisks� the target with a cell extension. This protein extension becomes a muscle-like headlock with foreign targets, wrapping around the target and pulling it in. Self-cells have a molecular protein on their surface that is specifically recognized by the macrophage to switch off the headlock, thus stopping engulfment and allowing self cells to coexist and move on.

The team found that both actin and non-muscle myosin were involved in the attack of foreign sheep cells due to recognition of the foreign protein CD47. Human red blood cells activated only actin, in a meet-and-greet maneuver. Macrophages, therefore, use a form of identification, much like police ask for a driver�s license at a traffic stop; however, not every cell tailed by antibodies is a danger to the body, so macrophages check for that marker, the protein CD47. If the latter is missing or even of the wrong species, the target is devoured.

Additional experiments also showed that protein CD47 affects its receptor, SIRP-alpha, which in turn inactivates the myosin by preventing the addition of a phosphate group to the brawny molecule. By shutting down a pulling protein needed to complete phagocytosis, CD47 prevents cells that belong from being eaten.

Via

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Protein in Embryonic Stem Cells Controls Malignant Tumor Cells

A protein that governs development of human embryonic stem cells (hESCs) also inhibits the growth and spread of malignant melanoma, the deadliest skin cancer, Northwestern University researchers have discovered. Metastatic melanoma, which develops from the transformation of skin pigment cells or melanocytes, has a death rate of more than 80 percent and a median survival of less than 7.5 months.

The Northwestern scientists, led by researcher Mary J. C. Hendrix, additionally found that the protein, called Lefty, prevents aggressive breast cancer cells from metastasizing. Death from metastatic breast cancer exceeded 40,000 in 2007, with over 180,000 new cases diagnosed in the United States.

Importantly, Lefty is secreted only in hESCs, and not in any other stem cell type tested – including stem cells isolated from amniotic fluid, cord blood or adult bone marrow – or placental cells.

Results of the study, described in an article in the March 3rd online version of The Proceedings of the National Academy of Sciences, build on an elegant body of research by the Hendrix lab to identify the genes and cellular pathways involved in cancer metastasis.

Hendrix is president and scientific director of the Children’s Memorial Research Center and professor in The Robert H. Lurie Comprehensive Cancer Center of Northwestern University and at Northwestern's Feinberg School of Medicine. Lynne-Marie Postovit, who was first author on the study and a post-doctoral trainee in the Hendrix lab, is currently an assistant professor at the University of Western Ontario, Canada.

Embryonic stem cells are pluripotent, meaning they can become any of 200-plus cell types in the adult body, depending on the signals they receive from their microenvironment (surrounding cells, tissues and vasculature). During cancer progression, malignant cells also receive and release signals from their microenvironment, cues that promote tumor growth and metastasis.

Groundbreaking work by Hendrix and colleagues is elucidating how, by becoming more like unspecialized stem cells, aggressive melanoma cells gain enhanced abilities to migrate, invade and metastasize while remaining virtually undetected by the immune system.

Hendrix and co-researchers previously demonstrated that a three-dimensional matrix conditioned by hESCs induced metastatic melanoma cells to revert to a normal, skin cell-like type with the ability to form colonies in the manner of hESCs (Postovit and Seftor et al, Stem Cells 24:501-505, 2006).

“This observation allowed us to appreciate the powerful influence of the hESC microenvironment on the reprogramming of metastatic melanoma cells,” Hendrix said.

In subsequent experiments, Hendrix, Postovit and co-researchers found that aggressive melanoma and breast cancer produce a “morphogenic” protein called Nodal, which is essential for human embryonic stem cell pluripotency (Topczewska et al, Nature Medicine 12:925-932, 2006). Other researchers have found that Nodal also is present in testicular cancer.

“Thus, Nodal may serve as a prognostic marker of aggressive behaviors in human cancers,” Hendrix said.

As described in the PNAS study, the Lefty protein inhibits production of Nodal and therefore plays a major role in embryonic cell differentiation and development – under normal circumstances.

Hendrix and colleagues discovered that metastatic tumor cells do not express Lefty, allowing them to overproduce Nodal in an unregulated manner.

However, when the group exposed metastatic tumor cells to the microenvironment of hESCs containing Lefty, they witnessed dramatically reduced Nodal expression (production) in these cancer cells together with decreased tumor cell growth and invasiveness and an increase in apoptosis, or programmed cell suicide.

Although exposure to a hESC microenvironment inhibited Nodal expression and tumor growth in both metastatic melanoma and breast cancer cells, the breast cancer cells underwent more complex reprogramming. Melanoma cells responded to the hESC-derived factors within three days, but breast cancer cells required two additional days to achieve the most significant reduction in Nodal.

This discrepancy is likely due to differences in signaling mechanisms between the two cell types. Yet, despite the inherent differences between melanoma cells and breast cancer cells, these divergent tumor types both underwent cell suicide following exposure to the hESC microenvironment.

“The remarkable similarity of the responses of the two tumor types is likely attributable to the commonality of plasticity (for example, the aberrant and unregulated expression of Nodal) that indiscriminately unifies highly aggressive cancer cells, regardless of their tissue of origin,” Hendrix said.

“Further, the tumor suppressive effects of the hECs microenvironment, by neutralizing the expression of Nodal in aggressive tumor cells, provide previously unexplored novel therapeutic modalities for cancer treatment,” Hendrix said.

However, while findings from the study suggest that hESC-derived Lefty may have potential to prevent metastasis, it is not the only tumor suppressive factor within the embryonic microenvironment.

Observations from the study highlight the potential utility of isolating factors within the hESC microenvironment responsible for influencing tumor cell fate and reversing the cancerous properties of metastatic tumor cells, such as melanoma and breast cancer.

Additional contributing authors on the study include N. Margaryan; E. Seftor; D. Kirschmann; D. Abbott; W. Wheaton; A. Lipavsky; and R. Seftor.

This study was supported by grants from the Illinois Regenerative Medicine Institute; the National Institutes of Health (CA50702 and CA121205); the Charlotte Geyer Foundation (to Dr. Hendrix); and a Canadian Institutes of Health Research Postdoctoral Fellowship (to Dr. Postovit).
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Genetic research unveils common origins for distinct clinical diagnoses

Researchers at Johns Hopkins have discovered that two clinically different inherited syndromes are in fact variations of the same disorder. Reporting in the April issue of Nature Genetics, the team suggests that at least for this class of disorders, the total number and “strength” of genetic alterations an individual carries throughout the genome can generate a range of symptoms wide enough to appear like different conditions.

“We’re finally beginning to blur the boundaries encompassing some of these diseases by showing that they share the same molecular underpinnings,” says Nicholas Katsanis, Ph.D., an associate professor of ophthalmology at the McKusick-Nathans Institute of Genetic Medicine at Hopkins. “This is important progress for several reasons. First, knowing what’s going on molecularly and being able to integrate rarer conditions under common mechanisms allows us to potentially help more people at once. Second, clinicians can finally begin to offer more accurate diagnoses based on what really matters: the state of affairs at the cellular/biochemical level. In time, this will empower genetic counseling and much improved patient management.”

Katsanis’s team studies Bardet-Biedl syndrome (BBS), a rare so-called ciliopathy that is characterized by a combination of vision loss, obesity, diabetes, extra digits and mental defects and caused by faulty cilia, tiny hairlike projections found on almost every cell of the body. Recently they started looking at another disease, Meckel-Gruber syndrome (MKS), which also shows cilia dysfunction but is clinically distinct from BBS and generally associated with prenatal or newborn death.

“While these two groups of patients exhibit such different clinical outcomes, the genes associated with both syndromes all seemed to be pointing at the same culprit: cilia,” says Katsanis. “So we wondered if BBS and MKS might actually represent different flavors of the same disease.”

The researchers sequenced the MKS genes from 200 BBS patients and found six families that, in addition to carrying BBS genetic mutations, also carried mutations in MKS genes. To figure out what, if any, effect these MKS mutations have on BBS, the team used a system they previously developed in zebrafish.

Knocking out BBS genes in zebrafish generates short fish with even shorter tails, among other malformations. Injecting normal BBS genes into these fish rescues them, resulting in normal looking fish.

The researchers reasoned that if MKS and BBS are indeed the same condition, then fish with the MKS genes knocked out should mimic the BBS knockout fish. They did. The team then went on to test mutant versions of MKS genes in BBS fish and found that three genes originally attributed to MKS do indeed cause BBS or render the BBS defects more pronounced, increasing the number of BBS genes to 14 in total.

“From a clinical perspective, these two syndromes look nothing alike, but molecularly, the genes involved clearly participate in the same fundamental processes,” says Katsanis. “This means that Meckel-Gruber and Bardet-Biedel actually represent a continuum of one disease. This never would have been discovered in the clinic-only molecular analysis can reveal these things.”

But what does this mean for clinicians and the diagnosis and treatment of these syndromes" Katsanis hopes that the growing body of molecular data will help move medicine away from symptom-defined syndromes, which can leave clinicians struggling with ambiguous diagnoses, to approaching disorders from a molecular standpoint. “We now have the possibility of merging several rare disorders,” he says. “And their gross sum now turns out to be fairly common; hopefully this will now put them on the radar for drug development and other therapies.”

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MIT researchers demonstrate protective role of microRNA

Snippets of genetic material that have been linked to cancer also play a critical role in normal embryonic development in mice, according to a new paper from MIT cancer biologists.

The work, reported in the March 7 issue of Cell, shows that a family of microRNAs--short strands of genetic material--protect mouse cells during development and allow them to grow normally. But that protective role could backfire: The researchers theorize that when these microRNAs become overactive, they can help keep alive cancer cells that should otherwise die--providing another reason to target microRNAs as a treatment for cancer.

Discovered only a decade ago, microRNAs bind to messenger RNAs (mRNAs), preventing them from delivering protein assembly instructions, thereby inhibiting gene expression. The details of how microRNAs act are not yet fully understood.

"The scientific community is busy trying to understand what specific biological functions these microRNAs affect," said Andrea Ventura, lead author of the paper and postdoctoral associate in the Koch Institute for Integrative Cancer Research at MIT (formerly known as the Center for Cancer Research).

Ventura--who works in the laboratory of Tyler Jacks, director of the Koch Institute--and her colleagues studied the function of a family of microRNAs known as the miR-17~92 cluster.

Previous research has shown that the miR-17~92 cluster is overactive in some cancers, especially those of the lungs and B cells.

To better understand these microRNAs' role in cancer, the researchers decided to study their normal function. Knocking out microRNA genes and observing the effects can offer clues into how microRNA helps promote cancer when overexpressed.

They found that when miR-17~92 was knocked out in mice, the animals died soon after birth, apparently because their lungs were too small. Also, their B cells, a type of immune cell, died in an early stage of cell development.

This suggests that miR-17~92 is critical to the normal development of lung cells and B cells. In B cells, these microRNAs are likely acting to promote cell survival by suppressing a gene that induces cell death, said Ventura.

"Understanding why these things are happening provides important insight into how microRNAs affect tumorigenesis," he said.

The researchers theorize that when miR-17~92 becomes overactive in cancer cells, it allows cells that should undergo programmed cell death to survive.

Blocking microRNAs that have become overactive holds promise as a potential cancer treatment. Research is now being done on molecules that prevent microRNAs from binding to their target mRNA.

More work needs to be done to make these inhibitors into stable and deliverable drugs, but Ventura said it's possible it could be done in the near future.

The exact genes targeted by miR-17~92 are not known, but one strong suspect is a gene called Bim, which promotes cell death. However, a single microRNA can have many targets, so it's likely there are other genes involved.

The researchers also studied the effects of knocking out two other microRNA clusters that are closely related to miR-17~92 but located elsewhere in the genome.

They found that if the other two microRNA clusters are knocked out but miR-17~92 remains intact, the mice develop normally. However, if miR-17~92 and one of these similar clusters are removed, the mice die before birth, suggesting there is some kind of synergistic effect between these microRNA families.

Other MIT authors of the paper are Amanda Young, graduate student in biology; Monte Winslow, postdoctoral fellow in the Center for Cancer Research (CCR); Laura Lintault, staff affiliate in the CCR; Alex Meissner, faculty member at the Broad Institute of MIT and Harvard; Jamie Newman, graduate student in biology; Denise Crowley, staff affiliate at the CCR; Rudolf Jaenisch, professor of biology and member of the Whitehead Institute for Biomedical Research; Phillip Sharp, MIT Institute Professor; and Jacks, who is also a professor of biology.

The research was funded by the National Institutes of Health and the National Cancer Institute.

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Device allows scientists to control gene activity across generations of cells

Just as cells inherit genes, they also inherit a set of instructions that tell genes when to become active, in which tissues and to what extent. Now, Rockefeller University researchers have built a device that, by allowing scientists to turn genes on and off in actively multiplying budding yeast cells, will help them figure out more precisely than before how genes and proteins interact with one another and how these interactions drive cellular functions.

�A slight disturbance in the abundance of a single protein can affect the functioning of a cell dramatically,�� says Gilles Charvin, a postdoc who works with both Eric Siggia, head of the Laboratory of Theoretical Condensed Matter Physics, and Frederick Cross, headof the Laboratory of Yeast Molecular Genetics. �So, we wanted to devise a way to supply a single cell with a controlled pulse of protein at any time and then see how the cell would respond,�� he says.

Although scientists have had the tools to track single cells and measure the protein levels within them, the new device allows scientists to track them for a longer period of time while not only monitoring but also controlling the activity of genes. The precision with which the device can track single cells also allows scientists to construct pedigrees, making it possible to compare gene activity from one cell to the next.

The device relies on electrovalves to control a flow of media, which travels through a tube and then diffuses across a porous membrane to reach the budding yeast cells. The cells are clamped between this membrane and a soft material, which forces them to bud horizontally without damage.

That was the major design hurdle,�� says Charvin. �To create a device in which cells don�t move, so that you can track hundreds of single cells for a long time � about eight rounds of cell division � which typically lasts 12 hours.��

In order to induce the activity of a gene, the researchers used inducer molecules that diffuse through the cell membrane and control DNA segments called promoters. The molecule�s presence silences the promoter, which silences the expression of the gene; the molecule�s absence, on the other hand, activates the promoter, which activates the gene to crank up the molecule�s production.

By exploiting this principle, the scientists showed that they could successfully turn specific genes on and off by controlling the flow of an inducer molecule called methionine. They observed that pulses as short as 10 minutes led to changes in protein levels that could be measured.

The group used this device to study the cell cycle by putting a gene that must be expressed for cells to divide under the control of the methionine promoter, and showed that budding yeast cells would stop and start dividing in perfect synchrony with alternating pulses of media that did and didn�t contain methionine. �Like slaves, the cells relied on the external pulse we gave them to figure out what to do next,� says Charvin. �We thought this was a pretty striking illustration of the capabilities of this device.�
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Evolving complexity out of 'junk DNA'

Vertebrates - animals such as humans that possess a backbone - are the most anatomically and genetically complex of all organisms, but explaining how they achieved this complexity has vexed scientists since the conception of evolutionary theory.

Alysha Heimberg of Dartmouth College and her colleagues showed that microRNAs, a class of tiny molecules only recently discovered residing within what has usually been considered ‘junk DNA’, are hugely diverse in even the most lowly of vertebrates, but relatively few are found in the genomes of our invertebrate relatives.

She explained: “There was an explosive increase in the number of new microRNAs added to the genome of vertebrates and this is unparalleled in evolutionary history.”

Co-author, Dr Philip Donoghue of Bristol University’s Department of Earth Sciences continued: “Most of these new genes are required for the growth of organs that are unique to vertebrates, such as the liver, pancreas and brain. Therefore, the origin of vertebrates and the origin of these genes is no coincidence.”

Dr Kevin Peterson of Dartmouth College said: “This study not only points the way to understanding the evolutionary origin of our own lineage, but it also helps us to understand how our own genome was assembled in deep time.”

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Genes linked with Lupus are revealed, giving hope for new treatments

Scientists have identified a number of genes involved in Lupus, a devastating autoimmune disease that affects around 50,000 people in the UK, in new research published today in the journal Nature Genetics.

In an international genetic study of more than 3,000 women, researchers found evidence of an association between Lupus (systemic lupus erythematosus or SLE) and mutations in several different genes.

The findings, by scientists from Imperial College London and institutions in the USA and Sweden, will enable researchers to investigate the specific pathways and precise molecular mechanisms involved in developing Lupus, potentially opening up options for new therapies. Lupus is a complex condition, mostly affecting women, which frequently causes skin rash, joint pains and malaise, and which can also lead to inflammation of the kidneys and other internal organs.

The scientists discovered the strongest associations with Lupus in three genes: ITGAM, PXK, and one mutation within a gene KIAA1542, a gene whose function is not definitely known.

The ITGAM gene provides code for a molecule involved in a system, known as the complement system, which forms part of the body's immune response. Complement is a series of proteins in the blood which is designed to stick to the surface of bacteria and bugs in order to enable them to be attacked by the immune system.

The discovery of variations in the ITGAM gene in people with Lupus supports the idea that abnormalities in the way complement and antibodies bind to immune cells play a key part in the disease. It is already known that people with Lupus often have low levels of complement in their blood.

The role of the molecules encoded by the PXK gene and KIAA1542 genes in Lupus is less easy to predict, and the discovery of their association is more surprising to the researchers, opening up new avenues of research into the disease.

Other genes, including LYN and BLK, also appear to be involved in Lupus. These genes affect the function of B cells, which play a key role in the production of antibodies. Autoantibodies, which attack the body's own proteins, contribute to the damage done to the body in Lupus.

The new research also confirms links identified in previous studies between Lupus, as well as other autoimmune diseases, and certain other genes.

Professor Timothy Vyse, a Wellcome Trust Senior Fellow from the Division of Medicine at Imperial College London, and one of the authors of the study, said: "Lupus is a complex disease, which is hard to diagnose, and it can cause many different and unpredictable problems for patients. Living with Lupus can be really tough. We currently can treat the disease by suppressing the immune system, but we urgently need to understand in much more detail what goes wrong with the immune system so that we can design better treatments. This study represents a milestone in progress towards unravelling the secrets of the disease.

"We are continuing to work on refining these genetic studies. Blood samples from patients with Lupus have helped us already and we are very grateful to those who have given us samples. We always need more samples and would like to hear from anyone with Lupus who would like to help us by giving blood samples for this important research," added Professor Vyse.

The researchers reached their conclusions after comparing the genetic makeup of 720 women of European descent with Lupus and 2,337 women without Lupus. They looked at mutations in the building blocks, called nucleotides, which make up DNA.

There are mutations in around one in every 600 nucleotides and the scientists examined over 317,000 how many of these mutations to find those specific to Lupus. These mutations are known as single-nucleotide polymorphisms.

The researchers confirmed their results by comparing another set of genetic data for 1,846 women with Lupus and 1,825 women without Lupus.

The study was carried out by researchers in the International SLE consortium (SLEGEN), which includes scientists from the USA, Sweden and the UK. It was supported by the Alliance for Lupus Research and the National Institutes of Health.

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'Telepathic' genes recognize similarities in each other

Genes have the ability to recognise similarities in each other from a distance, without any proteins or other biological molecules aiding the process, according to new research published this week in the Journal of Physical Chemistry B. This discovery could explain how similar genes find each other and group together in order to perform key processes involved in the evolution of species.

This new study shows that genes – which are parts of double-stranded DNA with a double-helix structure containing a pattern of chemical bases - can recognise other genes with a similar pattern of chemical bases.

This ability to seek each other out could be the key to how genes identify one another and align with each other in order to begin the process of ‘homologous recombination’ – whereby two double-helix DNA molecules come together, break open, swap a section of genetic information, and then close themselves up again.

Recombination is an important process which plays a key role in evolution and natural selection, and is also central to the body’s ability to repair damaged DNA. Before now, scientists have not known exactly how suitable pairs of genes find each other in order for this process to begin.

The authors of the new study carried out a series of experiments in order to test the theory, first developed in 2001 by two members of this team, that long pieces of identical double-stranded DNA could identify each other merely as a result of complementary patterns of electrical charges which they both carry. They wanted to verify that this could indeed occur without physical contact between the two molecules, or the facilitating presence of proteins.

Previous studies have suggested that proteins are involved in the recognition process when it occurs between short strands of DNA which only have about 10 pairs of chemical bases. This new research shows that much longer strands of DNA with hundreds of pairs of chemical bases seem able to recognise each other as a whole without protein involvement. According to the theory, this recognition mechanism is stronger the longer the genes are.

The researchers observed the behaviour of fluorescently tagged DNA molecules in a pure solution. They found that DNA molecules with identical patterns of chemical bases were approximately twice as likely to gather together than DNA molecules with different sequences.

Professor Alexei Kornyshev from Imperial College London, one of the study’s authors, explains the significance of the team’s results: “Seeing these identical DNA molecules seeking each other out in a crowd, without any external help, is very exciting indeed. This could provide a driving force for similar genes to begin the complex process of recombination without the help of proteins or other biological factors. Our team’s experimental results seem to support these expectations.”

Understanding the precise mechanism of the primary recognition stage of genetic recombination may shed light on how to avoid or minimise recombination errors in evolution, natural selection and DNA repair. This is important because such errors are believed to cause a number of genetically determined diseases including cancers and some forms of Alzheimer’s, as well as contributing to ageing. Understanding this mechanism is also essential for refining precise artificial recombination techniques for biotechnologies and gene therapies of the future.

The team is now working on a set of further experiments to determine exactly how these interactions work, including the predicted length dependence. In addition, further studies are needed to ascertain whether this interaction, discovered in a test tube, occurs in the highly complex environment of a living cell.

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Protein discovered that prevents HIV from spreading

n a study that could open up the field of virology to an entirely new suite of possibilities and that paves the way for future drug research, scientists at Rockefeller University and the Aaron Diamond AIDS Research Center have pinned down a molecule on the surface of human cells that helps keep particles of mutant strains of HIV from spreading. Rather than floating off to infect more cells, the protein contains the virus particles by keeping them attached to the parent cell�s outer membrane, as if stuck there with glue.

Two years ago, Paul Bieniasz � head of the Laboratory of Retrovirology and ADARC scientist �discovered that normal HIV-1 particles are able to extricate themselves from the sticky membranesurface using a protein called Vpu. Bieniasz has been searching for the source of the glue itself ever since. Now, in an advanced online publication in Nature, he and his colleagues report that they found it: a protein they dubbed �tetherin� for its ability to keep viruses tied to a cell.

�All we knew when we started this two and a half years ago was that a virus lacking Vpu was released less efficiently from cells.� Bieniasz says. �And we had some electron micrographs that showed virus particles stuck there on the surface and clustered inside cells.� Once they started looking carefully at the reasons behind this, they found an antiviral mechanism keeping the HIV-1 mutant particles tethered to the cell. And it wasn�t just HIV � the glue appeared to interfere with the spread of other membrane-encapsulated (or �enveloped�) viruses, too.

To track down the cause of stickiness � and the likely reason HIV evolved Vpu � Bieniasz and his team looked at gene activity across all known human genes, making comparisons between cells that require Vpu for HIV-1 release and those that don�t. Ultimately, they narrowed it down to one very likely candidate. And the candidate, the tetherin protein, passed all the tests the researchers threw at it: When Vpu was not present but tetherin was, large numbers of virus particles piled up on the cell surface. When tetherin was missing, however, even the Vpu-deficient viruses were able to escape.

�We�ve discovered a new way that cells defend themselves against viruses,� Bieniasz says. �I think this will open up a new area of study in virology: how this protein antagonizes other viruses, and how viruses learn to get around it.� Going forward, his lab will focus on how broad tetherin�s antiviral activity is, and whether variations of it exist that might confer additional immunity or sensitivity to HIV and other viruses. And, he notes, if drug researchers are able to interfere with the interaction between tetherin and Vpu, their newly discovered protein might even provide a potential therapeutic target.

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Mammalian protein plays unexpected role in cell division, and perhaps cancer

The French Nobel laureate Jacques Monod famously said, �What�s true for E.coli is true for an elephant.� With this in mind, researchers at Rockefeller University set out to determine the function of Tel2, a protein originally found in yeast where it maintains the length of chromosome tips called telomeres. But one experiment after another informed the group that Tel2 in humans plays an altogether different role.

Researchers led by Titia de Lange, head of the Laboratory of Cell Biology and Genetics, now reveal that mammalian Tel2 stabilizes a family of six proteins called PIKKs, enzymes that catalyze the �stop� or �go ahead� signals at certain checkpoints in the cell cycle. Without the coordinated activity of these proteins, cells either stop dividing and ultimately perish or accumulate mutations and form tumors. This research � and its surprising conclusion � appears in the December 28 issue of Cell.

When de Lange and Hiroyuki Takai, a postdoc in her lab, found that Tel2 had no obvious function in mammalian telomeres, they almost dropped the project. But then Takai noticed that cells without Tel2 were unable to detect damage in their DNA, a function carried out by two members of the PIKK family: ATM and ATR. �So Hiro decided to measure their levels in these Tel2 knockout mice and saw that within three or four days the two proteins were gone,� says de Lange, who is also Leon Hess Professor at Rockefeller. After measuring the four other PIKKs, de Lange and Takai found that only PIKKs disappeared in these cells, suggesting that Tel2 specifically targets this family of signal transducers.

Takai and de Lange determined that Tel2 prevented the degradation of these proteins by using a laborious, �time-honored� technique called pulse-chase labeling. With this technique, they found that cells without Tel2 were able to synthesize the six proteins but were unable to keep them around. Tel2 doesn�t affect their synthesis but their stability. The group further showed that Tel2 stabilizes each of these six PIKKs by binding to a region common to all of them.

In addition to ATR and ATM, the PIKK family includes SMG1, TRRAP, DNA-PKcs and mTOR � �all kinases that regulate central pathways of enormous importance to human disease,� says de Lange. In particular, tumor cells depend on mTOR to survive and to a large extent ATR and ATM; for some time now, mTOR has been a target in clinical trials to combat cancer.

�We are excited about the possibility of using our findings to manipulate PIKKs in tumor cells and thereby kill them,� says de Lange. �As always with new approaches to cancer therapy, the challenge will be in figuring out how to avoid harming cells that are healthy.�

De Lange, who is usually funded for her work on telomeres, was supported in large part by grants from the Breast Cancer Research Foundation and the National Cancer Institute at the National Institutes of Health.
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Cell-to-Cell Communication in Bacteria

The research in my laboratory focuses on the molecular mechanisms that bacteria use for intercellular communication. Our goal is to understand how bacteria detect multiple environmental cues, and how the integration and processing of this information results in the precise regulation of gene expression.

The bacterial communication phenomenon that we study is called quorum sensing, which is a process that allows bacteria to communicate using secreted chemical signaling molecules called autoinducers. This process enables a population of bacteria to collectively regulate gene expression and, therefore, behavior. In quorum sensing, bacteria assess their population density by detecting the concentration of a particular autoinducer, which is correlated with cell density. This “census-taking” enables the group to express specific genes only at particular population densities. Quorum sensing is widespread; it occurs in numerous Gram-negative and Gram-positive bacteria. In general, processes controlled by quorum sensing are ones that are unproductive when undertaken by an individual bacterium but become effective when undertaken by the group. For example, quorum sensing controls bioluminescence, secretion of virulence factors, sporulation, and conjugation. Thus, quorum sensing is a mechanism that allows bacteria to function as multi-cellular organisms.

We have shown that the model luminous bacterium Vibrio harveyi and the related pathogen Vibrio cholerae each produce two different autoinducers, called AI-1 and AI-2, each of which is detected by its own sensor protein. Both sensors transduce information to a shared integrator protein to control the output, light emission in V. harveyi and virulence in V. cholerae. We have cloned the genes for signal production, detection and response in both species and have shown that the mechanism of signal relay is a phosphorylation/dephosphorylation cascade (see figure). Our recent studies combining genetics and bioinformatics (in collaboration with the Wingreen lab) show that the small RNA chaperone protein Hfq, together with multiple small regulatory RNAs (sRNAs), act at the center of these quorum sensing cascades. They function as an ultrasensitive regulatory switch that controls the critical transition into and out of quorum sensing mode.

V. harveyi and V. cholerae use the AI-1 quorum sensing circuit for intra-species communication and the AI-2 quorum sensing circuit for inter-species communication. To investigate the mechanism of AI-2 signaling, we constructed mutants and cloned the gene responsible for AI-2 production from several bacteria. The gene we identified in each case is highly homologous, and we named it luxS. We found that luxS homologues and AI-2 production are widespread in the bacterial world suggesting that communication via an AI-2 signal response system could be a common mechanism that bacteria employ for inter-species interaction in natural environments. We determined the biosynthetic pathway for AI-2 production as well as the AI-2 identity by solving the crystal structures of the V. harveyi and S. typhimurium sensor proteins in complex with their cognate AI-2 signals. The structural work was performed in collaboration with the Hughson lab. The V. harveyi AI-2 is a furanosylborate diester. Finding boron in the active molecule was surprising because boron, while widely available in nature has almost no known role in biology. The S. typhimurium crystal showed that its receptor binds a chemically distinct AI-2 that lacks borate. Importantly, the active signal molecules spontaneously inter-convert upon release from their respective receptors, revealing a surprising level of sophistication in the chemical lexicon used by bacteria for inter-species cell-cell communication.

Finally, we are focused on developing molecules that are structurally related to AI-2. Such molecules have potential use as anti-microbial drugs aimed at bacteria that use AI-2 quorum sensing to control virulence. Similarly, the biosynthetic enzymes involoved in AI-2 production and the AI-2 detection apparatuses are viewed as potential targets for novel anti-microbial drug design.

Source:http://www.molbio2.princeton.edu

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