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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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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Biologists identify key protein in cell’s 'self-eating' function

Molecular biologists at the University of California, San Diego have found one piece of the complex puzzle of autophagy, the process of “self-eating” performed by all eukaryotic cells -- cells with a nucleus -- to keep themselves healthy.

Their finding, published in the March 11 issue of the journal Developmental Cell, is important because it allows scientists to control this one aspect of cellular autophagy, and may lead to the ability to control other selective “self-eating” processes. This, in turn, could help illuminate autophagy’s role in aging, immunity, neurodegeneration and cancer.

All eukaryotic cells dispose of bacteria, viruses, damaged organelles and other non-essential components through this self-eating process. A part of the cell called the lysosome engulfs and degrades subcellular detritus. The ability of cells to recycle and reuse the cellular raw materials, as well as to “re-model” themselves in response to changing conditions, allows them to adapt and survive.

Autophagy was first described about 40 years ago, but has recently become a topic of great interest in cell biology because it is linked to cell growth, development aging and homeostasis -- helping cells to maintain a balance among synthesis, degradation and recycling.

The UC San Diego researchers report in their paper that they identified a novel protein called Atg30 (one of 31 required for autophagy-related processes) from the yeast Pichia pastoris, that controls the degradation of a sub-compartment of cells, the peroxisomes.

Peroxisomes generate and dispose of harmful peroxides that are by-products of oxidative chemical reactions.

Different organelles within the cell are degraded by lysosomes when the organelles are damaged or not necessary, said Jean-Claude Farré, the biologist who identified Atg30. The team is investigating peroxisomes, and working to understand how and why they are selected by the lysosome for degradation.

What the biologists found, he said, is that “this new protein can mediate peroxisome selection during pexophagy – that is, it is necessary for pexophagy, but not for other autophagy-related processes.”

Suresh Subramani, a professor of biology who headed the team, said they have established that Atg30 is a “key player” in the selection of peroxisomes for delivery to “the autophagy machinery” for re-cycling.

“For the first time, we can use a protein to control the process,” Subramani said. “It’s an important step in understanding the workings of cells.”

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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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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.�
SOURCE

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