Two biologists at the University of California, San Diego have discovered the first of a new class of cellular motor proteins that “rewind” sections of the double-stranded DNA molecule that become unwound, like the tangled ribbons from a cassette tape, in “bubbles” that prevent critical genes from being expressed.

“When your DNA gets stuck in the unwound position, your cells are in big trouble, and in humans, that ultimately leads to death” said Jim Kadonaga, a professor of biology at UCSD who headed the study.  “What we discovered is the enzyme that fixes this problem.”

The discovery represents the first time scientists have identified a motor protein specifically designed to prevent the accumulation of bubbles of unwound DNA, which occurs when DNA strands become improperly unwound in certain locations along the molecule.

The UCSD researchers’ findings, detailed in the October 31 issue of Science, are also important because they provide biomedical scientists with a greater understanding of the molecular mechanisms that lead to a rare genetic disorder called Schimke immuno-osseous dysplasia.  The discovery will eventually allow medical researchers to design future treatments for this devastating genetic disorder, which causes strokes, congestive heart failure, kidney failure and death in young children.

“We knew this particular protein caused this disease before we started the study,” said Kadonaga.  “That’s why we investigated it.  We just didn’t know what it did.”

What this protein, called HARP for HepA-related protein, did astounded Kadonaga and Timur Yusufzai, a postdoctoral fellow working in his laboratory.  The two molecular biologists initially discovered that this motor protein burns energy in the same way as enzymes called helicases and, like helicases, attached to the dividing sections of DNA.  But while helicases use their energy to separate two annealed nucleic acid strands—such as two strands of DNA, two strands of RNA or the strands of a RNA-DNA hybrid— the scientists found to their surprise that this protein did the opposite; that is, it rewinds sections of defective DNA and thus seals the two strands together again.

As a consequence, the UCSD biologists termed their new enzyme activity an “annealing helicase.”

“We didn’t even consider the idea of annealing helicases before this study started,” said Kadonaga.  “It didn’t occur to us that such enzymes even existed.  In fact, we never knew until now what happened to DNA when it got stuck in the unwound position.”

Now scientists who study the action of helicases on DNA and RNA have an entirely new class of proteins to investigate.

“This will open up a whole new area of study,” said Kadonaga.  “There are very few enzymes known that alter DNA structure.  And we’ve discovered an entirely new one.  This was not expected to happen in the year 2008.  We should have found them all by now.”

“I believe it’s going to go beyond DNA.  Just as there are DNA-DNA helicases, there are RNA-DNA helicases and RNA-RNA helicases.  So it doesn’t take a lot of imagination to foresee that there are probably going to be RNA-DNA annealing helicases and RNA-RNA annealing helicases.  The field potentially can be fairly large.  And as more and more people discover additional annealing helicases, this field will expand.”

Kadonaga and Yusufzai are already searching for more annealing helicases, but they also plan to continue their studies of HARP.

“First, what we want to do is find more of these proteins, so we’re looking for more right now,” said Kadonaga.  “We also want to see what other specific processes are affected by this particular protein, HARP, in the cell.”

In the nucleus, DNA wraps around histone proteins, which pack the DNA and makes it less accessible for transcription. Many transcriptional activators promote histone acetylation, which opens the chromatin structure and helps recruit transcription machinery to the newly accessible genes. Conversely, some gene repressors promote deacetylation of histones. Because drug dependence is mediated partly by changes in gene expression, inhibitors of histone acetylation and deacetylation might prevent the development of drug dependence. Romieuet al. support this hypothesis by showing that administering histone deacetylase inhibitors shortly before giving rats access to cocaine reduced cocaine self-administration and decreased the number of times a rat poked its nose in a hole to receive a dose of cocaine. When rats receive cocaine daily, their response to a dose increases over time. This increased responsiveness, called sensitization, is thought to promote dependence. Cocaine sensitization is prevented by histone deacetylase inhibitors, suggesting inhibitors may effectively reduce dependence.

The complete genetic blueprint for lethal pancreatic cancer and brain cancer was deciphered by a team at the Johns Hopkins Kimmel Cancer Center.

The studies, led by the same group who completed maps of the breast cancer and colorectal cancer genomes in 2007, are reported in two articles in the Sept. 5, 2008, issue of Science Express.

Believed to be the most comprehensive result to date for any tumor type, the new map evaluated mutations in virtually all known human protein-encoding genes, comprised of more than 20,000 genes, in 24 pancreatic cancers and 22 brain cancers.

A core set of regulatory gene processes and pathways, about a dozen for each tumor type, were found to be altered in the majority of tumors studied by the researchers. In pancreatic cancer, these 12 pathways, including those linked to DNA damage control, cell maturation, and tumor invasion, were altered in 67 percent to 100 percent of tumors.

“This perspective changes the way we think about solid tumors and their management, because drugs or other agents that target the physiologic effects of these pathways, rather than individual gene components, are likely to be the most useful approach for developing new therapies,” says Bert Vogelstein, M.D., co-director of the Ludwig Center at Johns Hopkins and a Howard Hughes Medical Institute investigator.

In addition to the pathway discoveries, a number of individual mutated genes were identified, including 83 cancer genes in pancreatic cancer and 42 in the most lethal form of brain cancer, glioblastoma multiforme (GBM). Additionally, 70 genes that were dramatically overexpressed in either cancer encode proteins that are on the surface of cells or secreted, making them potential diagnostic and screening targets.

One gene, isocitrate dehydrogenase 1 (IDH1), was found to be frequently mutated in a subset of GBM brain cancers. The mutations were significantly more common in young GBM patients, and were associated with improved survival. IDH1 mutations were also found in nearly all cases of secondary GBMs (cancers that progress from pre-existing lower grade tumors), raising the possibility that this mutation may be a useful marker for identifying which low-grade brain tumors are most likely to develop into the lethal GBMs.

“Patients with IDH1 mutations seem to be different from other patients with GBM, both clinically and biologically,” says Victor Velculescu, M.D., Ph.D., associate professor of oncology. “It is conceivable that these patients will ultimately benefit from different treatments, potentially by targeting IDH1.”

“The landscape of human cancers is clearly more complex than has been previously appreciated. Fighting it is going to be more of a guerilla war than a conventional one because there are dozens of mutated genes in each tumor,” says Kenneth W. Kinzler, Ph.D., co-director of the Ludwig Center at Johns Hopkins and professor of oncology. “Individually, these mutations don’t seem formidable. But working together, they form an enemy that will require us to develop novel strategies to combat them, and the best long-term strategy may be early detection of tumors, when the number of guerilla warriors is still small and more easily handled.”

To make their findings, the investigators integrated several methods of genetic analysis. They used high-density microarrays to identify copy number alterations (amplifications and deletions) and next-generation sequencing technologies to evaluate gene expression. They also developed novel statistical algorithms to integrate these complementary genetic analyses, as well as techniques to separate alterations likely to contribute to cancer initiation and progression from so-called passenger mutations, which accumulate harmlessly during cancer development.

Each project cost more than $4 million, with lead funding for the Goldman Pancreatic Cancer Genome Initiative coming from the Sol Goldman Charitable Trust and Lillian Goldman Charitable Trust. The Virginia and D. K. Ludwig Foundation provided lead funding for the brain cancer project. The Ludwig Brain Tumor Initiative represents the first formal collaboration of Ludwig Centers established by the Ludwig Fund in 2006.

This year an estimated 38,000 people will develop pancreatic cancer in the US, with overall survival rates less than 5 percent. Although fewer patients are diagnosed with brain cancers (approximately 20,000 cases per year in the United States), the results are equally catastrophic. “The main reasons we chose to focus on these cancers is because they are so deadly and have such limited treatment options. What we learn about these tumors may lead to improved diagnostic measures or therapies in the future,” says Ralph Hruban, M.D., Director of the Sol Goldman Pancreatic Cancer Research Center at Johns Hopkins.

In a surprising reversal of conventional wisdom, a DNA-based study has revealed that the last of the woolly mammoths—which lived between 40,000 and 4,000 years ago—had roots that were exclusively North American.The research, which appears in the September issue of Current Biology, is expected to cause some controversy within the paleontological community.

“Scientists have always thought that because mammoths roamed such a huge territory—from Western Europe to Central North America—that North American woolly mammoths were a sideshow of no particular significance to the evolution of the species,” said Hendrik Poinar, associate professor in the departments of Anthropology, and Pathology & Molecular Medicine at McMaster University.

Poinar and Régis Debruyne, a postdoctoral research fellow in Poinar’s lab, spent the last three years collecting and sampling mammoths over much of their former range in Siberia and North America, extracting DNA and meticulously piecing together, comparing and overlapping hundreds of mammoth specimen using the second largest ancient DNA dataset available.

“Migrations over Beringia [the land bridge that once spanned the Bering Strait] were rare; it served as a filter to keep eastern and western groups or populations of woollies apart, says Poinar. “However, it now appears that mammoths established themselves in North America much earlier than presumed, then migrated back to Siberia, and eventually replaced all pre-existing haplotypes of mammoths.”

“Small-scale population replacements, as we call them, are not a rare phenomenon within species, but ones occurring on a continental scale certainly are,” says Ross MacPhee, curator of mammalogy at the American Museum of Natural History, and one of the researchers on the study. “We never expected that there might have been a complete overturn in woolly mammoths, but this is the sort of discoveries that are being made using ancient DNA. Bones and teeth are not always sensitive guides.”

“Like paleontologists, molecular biologists have long been operating under a geographic bias,” says Debruyne. “For more than a century, any discussion on the woolly mammoth has primarily focused on the well-studied Eurasian mammoths. Little attention was dedicated to the North American samples, and it was generally assumed their contribution to the evolutionary history of the species was negligible. This study certainly proves otherwise.”

The origin of mammoths is controversial in itself. Some scientists believe that the first proto-mammoths arose in Africa about seven-million years ago in concert with ancestors of the Asian elephant. Around five to six million years ago, an early mammoth species migrated north into China, Siberia and, eventually, North America. This early dispersal into North America gave rise to a new mammoth known as the Columbian mammoth. Much later, back in Siberia, a cold-adapted form—the woolly mammoth—evolved and eventually crossed over the Beringian land bridge into present-day Alaska and the Yukon.

What happened next, says Poinar, is a mystery: The Siberian genetic forms began to disappear and were replaced by North American migrants.

“The study of evolution is an evolution in itself,” says Poinar. “This latest research shows we’re drilling down and getting a closer and better understanding of the origins of life on our planet.”

Out of the 3 billion genetic letters that spell out the human genome, Yale scientists have found a handful that may have contributed to the evolutionary changes in human limbs that enabled us to manipulate tools and walk upright.

Results from a comparative analysis of the human, chimpanzee, rhesus macaque and other genomes reported in the journal Science suggest our evolution may have been driven not only by sequence changes in genes, but by changes in areas of the genome once thought of as “junk DNA.”

Those changes activated genes in primordial thumb and big toe in a developing mouse embryo, the researchers found.

“Our study identifies a potential genetic contributor to fundamental morphological differences between humans and apes,” said James Noonan, Assistant Professor of Genetics in the Yale University School of Medicine and the senior author of the study.

Researchers have long suspected changes in gene expression contributed to human evolution, but this had been difficult to study until recently because most of the sequences that control genes had not been identified. In the last several years, scientists have discovered that non-coding regions of the genome, far from being junk, contain thousands of regulatory elements that act as genetic “switches” to turn genes on or off.

An indication of their biological importance, many of these non-coding sequences have remained similar, or “conserved,” even across distantly related vertebrate species such as chickens and humans. Recent functional studies suggest some of these “conserved non-coding sequences” control the genes that direct human development.

In collaboration with scientists at Lawrence Berkeley National Laboratory in California, the Genome Institute of Singapore, and the Medical Research Council in the United Kingdom, Noonan searched the vast non-coding regions of the human genome to identify gene regulatory sequences whose function may have changed during the evolution of humans from our ape-like ancestors.

Noonan and his colleagues looked for sequences with more base pairs in humans than in other primates. The most rapidly evolving sequence they identified, termed HACNS1, is highly conserved among vertebrate species but has accumulated variations in 16 base pairs since the divergence of humans and chimpanzees some 6 million years ago. This was especially surprising, as the human and chimpanzee genomes are extremely similar overall, Noonan said.

Using mouse embryos, Noonan and his collaborators examined how HACNS1 and its related sequences in chimpanzee and rhesus monkey regulated gene expression during development. The human sequence activated genes in the developing mouse limbs, in contrast to the chimpanzee and rhesus sequences. Most intriguing for human evolution, the human sequence drove expression at the base of the primordial thumb in the forelimb and the great toe in the hind limb. The results provided tantalizing, but researchers say preliminary, evidence that the functional changes in HACNS1 may have contributed to adaptations in the human ankle, foot, thumb and wrist– critical advantages that underlie the evolutionary success of our species.

However, Noonan stressed that it is still unknown whether HACNS1 causes changes in gene expression in human limb development or whether HACNS1 would create human-like limb development if introduced directly into the genome of a mouse.

“The long-term goal is to find many sequences like this and use the mouse to model their effects on the evolution of human development,” Noonan said.

Researchers at the University of Cincinnati (UC) are looking for ways to reduce or prevent heart damage by starting where the problem often begins: in the genes.

Following a heart attack, cells die, causing lasting damage to the heart.

Keith Jones, PhD, a researcher in the department of pharmacology and cell biophysics, and colleagues are trying to reduce post-heart attack damage by studying the way cells die in the heart—a process controlled by transcription factors.

Transcription factors are proteins that bind to specific parts of DNA and are part of a system that controls the transfer of genetic information from DNA to RNA and then to protein.  Transfer of genetic information also plays a role in controlling the cycle of cells—from cell growth to cell death.

“We call it ‘gene regulatory therapy,’” says Jones.

So far, studies have identified the role for an important group of interacting transcription factors and the genes they regulate to determine whether cells in the heart survive or die after blood flow restriction occurs.

Often, scientists use virus-like mechanisms to transfer DNA and other nucleic acids inside the body.

The “virus” takes over other healthy cells by injecting them with its DNA.  The cells, then transformed, begin reproducing the virus’ DNA.  Eventually they swell and burst, sending multiple replicas of the virus out to conquer other cells and repeat the process.

Now, UC researchers are further investigating new, non-viral delivery mechanisms for this transfer of DNA.

“We can use non-viral delivery vehicles to transfer nucleic acids, including transcription factor decoys, to repress activation of specific transcription factors in the heart,” Jones says, adding that the researchers have made this successfully work within live animal models.  “This means we can block the activity of most transcription factors in the heart without having to make genetically engineered mice.”

Jones will be presenting these results at the International Society for Heart Research in Cincinnati, June 17-20.

He says this delivery mechanism involves flooding the cells with “decoys” which trick the transcription factors into binding to the decoys rather than to target genes, preventing them from activating those genes.

“We can use this technology to identify the target genes and then investigate the action of these genes in the biological process,” Jones says.

He says that this delivery has limitations and advantages.

“It can be used to block a factor at any point in time and is reversible,” he says.  “However, right now, a specific delivery route must be used to target the tissue or cell.”

Jones and other researchers are hoping that this new technology will allow them to directly address the effects of gene regulation in disease, as opposed to using classical drugs that treat symptoms or have significant adverse outcomes.

“So far, this seems to cause no adverse effects in animals,” he says.  “We are hopeful and are working toward pre-clinical studies.”

For the first time, researchers at Delft University of Technology have witnessed the spontaneous repair of damage to DNA molecules in real time. They observed this at the level of a single DNA molecule. Insight into this type of repair mechanism is essential as errors in this process can lead to the development of cancerous cells. Researchers from the Kavli Institute of Nanoscience Delft are to publish an article on this in the leading scientific journal Molecular Cell.

Cells have mechanisms for repairing the continuous accidental damage occurring in DNA. These damages can vary from a change to a single part of the DNA to a total break in the DNA structure. These breaks can, for instance, be caused by ultraviolet light or X-rays, but also occur during cell division, when DNA molecules split and form two new DNA molecules. If this type of break is not properly repaired it can be highly dangerous to the functioning of the cell and lead to the creation of a cancerous cell.

One major DNA-repair mechanism involved in repairing these breaks is known as homologous recombination. This mechanism has been observed for the first time by Delft University of Technology researchers in real time and at the level of a single DNA molecule.

To observe this, a DNA molecule is stretched between a magnetic bead and a glass surface. A force is exerted on the magnetic bead using a magnetic field, enabling researchers to pull and rotate a single DNA molecule in a controlled fashion. As the position of the bead changes when the DNA molecule is repaired, researchers are able to observe the repair process in detail.

University of Illinois researchers have developed a technique for imaging cells under an electron microscope that yields a sharper image of the structure of chromatin, the tightly wound bundle of genetic material and proteins that makes up the chromosomes. The findings appeared in Nature Methods.

Scientists have known for more than a century that proteins, such as histones, aid in packing DNA into the nucleus of a cell.  Human cells contain 2 to 3 meters of DNA, which must be kinked and coiled enough to fit into a region 1/10 the width of a human hair.

Despite the use of powerful, high-resolution imaging techniques such as electron microscopy, the mechanism by which this chromatin packing occurs remains a mystery.  The densely coiled chromatin fibers are very difficult to visualize, and little is known about how they condense during cell division, or unwind to allow gene expression.

In developing their method, the Illinois team tackled a key difficulty in imaging cells using electron microscopy.  Traditional studies “fix” the cells with potent chemicals (called fixatives) to preserve their structure for viewing under a microscope.  But standard fixation methods interfere with another step in the imaging process: the use of tagged antibodies to label key components of the cells.

These antibodies, which target and latch on to specific proteins in the cell, can be tagged with fluorescent labels for detection in light microscopy, or with metal particles (gold, in this case) for electron microscopy.

“If you fix the cells first, you have a dramatic drop in the efficiency of these immunochemical reactions,” said Igor Kireev, a visiting scientist in the department of cell and developmental biology and lead author of the paper.  Electron microscopy image Click photo to enlarge Image courtesy of Andrew Belmont and Igor Kireev The new technique exposes living cells to labeled antibodies, an approach that yields a much stronger signal for electron microscopy.

“And if your target is inside the condensed chromatin, the antibodies have no way to penetrate.”

Instead of fixing the cells before staining with antibodies, the researchers first exposed living animal cells to the labeled antibodies.  This allowed the antibodies to penetrate more deeply into the chromatin structure, and boosted the number of gold particles adhering to regions of interest.  The signal was enhanced by adding a silver solution that precipitated (solidified) upon contact with the gold.

“We are interested in chromatin structure, so our targets are mostly chromatin-bound proteins,” Kireev said.

The researchers had inserted several copies of a bacterial DNA, called the Lac operator, into the chromosomes.  A bacterial protein, the Lac repressor, recognizes and binds to the Lac operator in living cells.

The researchers combined a Lac repressor protein with another protein that fluoresces green under blue light.  This engineered protein adhered to the chromosomes in regions containing the Lac operator sequences.  Under blue light, these regions fluoresced.  A gold-tagged antibody targeted against green fluorescent protein (GFP) was then microinjected into the nucleus of a living cell, which added a metallic signal that could be boosted with silver.

“All this combined gives us a much better signal, a much stronger signal, with the very best structural preservation,” Kireev said.

The fluorescing protein helped the researchers find the regions of interest in the cells.  These areas were then “immunogold” labeled and targeted for electron microscopy.

In the resulting micrographs the researchers saw enhanced staining of the chromosomes.

“We can now apply this same live-cell labeling method to study at high resolution many different GFP-tagged proteins in the cell cytoplasm or nucleus,” said Andrew Belmont, a professor of cell and developmental biology and senior author of the paper.

“In trying to understand chromosomes, people have largely been limited to low resolution visualization of specific chromosomal proteins using light microscopy,” Belmost said.  “This meant everyone has had to do a lot of guessing of how things are put together, leading in many cases to vague, cartoon models of what are likely to be complicated chromosomal structures carrying out DNA functions such as replication and transcription.”

“Now we hope we can simply look and see the real structure using the more than 10-fold higher resolution of electron microscopy,” Belmont said.  “We are really excited to see what we will find using our new method”

(LA JOLLA, CA) Salk researchers zoom in on genome-wide DNA methylation and transcriptomes at single base resolution. Until quite recently, the chemical marks littering the DNA inside our cells like trees dotting a landscape could only be studied one gene at a time.  But new high-throughput DNA sequencing technology has enabled researchers at the Salk Institute for Biological Studies to map the precise position of these individual DNA modifications throughout the genome of the plant Arabidopsis thaliana, and chart its effect on the activity of any of Arabidopsis’ roughly 26,000 genes.

“For a long time the prevailing view held that individual modifications are not critical,” says Joseph Ecker, Ph.D., a professor in the Plant Biology laboratory and director of the Salk Institute Genomic Analysis Laboratory.  “The genomes of higher eukaryotes are peppered with modifications but unless you can take a detailed look at a large scale there is no way of knowing whether a particular mark is critical or not.”

The Salk study, which appears today in the online issue of Cell, paints a detailed picture of a dynamic and ever-changing, yet highly controlled, epigenome, the layer of genetic control beyond the regulation inherent in the sequence of the genes themselves.

Being able to study the epigenome in great detail and in its entirety will provide researchers with a better understanding of plant productivity and stress resistance, the dynamics of the human genome, stem cells’ capacity to self-renew and how epigenetic factors contribute to the development of tumors and disease.

Discoveries in recent years made it increasingly clear that there is far more to genetics than the sequence of building blocks that make up our genes.  Adding molecules such as methyl groups to the backbone of DNA without altering the letters of the DNA alphabet can change how genes interact with the cell’s transcribing machinery and hand cells an additional tool to fine-tune gene expression.

“The goal of our study was to integrate multiple levels of epigenetic information since we still have a very poor understanding of the genome-wide regulation of methylation and its effect on the transcriptome,” explains postdoctoral researcher and co-first author Ryan Lister, Ph.D.

The transcriptome encompasses all RNA copies or transcripts made from DNA.  The bulk of transcripts consists of messenger RNAs, or mRNAs, that serve as templates for the manufacture of proteins but also includes regulatory small RNAs, or smRNAs.  The latter wield their power over gene expression by literally cutting short the lives of mRNAs or tagging specific sequences in the genome for methylation.

But before Lister could start to unravel the multiple layers of epigenetic regulation that control gene expression, he had to pioneer new technologies that allowed him to look at genome-wide methylation at single-base resolution and to sequence the complete transcriptome within a reasonable timeframe.

Collaborating scientists at the ARC Centre of Excellence in Plant Energy Biology at the University of Western Australia in Perth developed a powerful, web-based genome browser, which played a crucial role in unlocking the information hidden in the massive datasets.

Cells employ a whole army of enzymes that add methyl groups at specific sites, maintain established patterns or remove undesirable methyl groups.  When Lister and his colleagues compared normal cells with cells lacking different combination of enzymes they discovered that cells put a lot of effort in keeping certain areas of the genome methylation-free.

On the flipside, the Salk researchers found that when they knocked out a whole class of methylases, a different type of methylase would step into the breach for the missing ones.  This finding is relevant for a new class of cancer drugs that work by changing the methylation pattern in tumor cells.

“You might succeed in removing one type of methylation but end up with increasing a different type,” says Ecker.  “But very soon we will be able to look and see what kind of compensatory changes are happening and avoid unintended consequences.”

Previous studies had found that a subset of smRNAs could direct methylation enzymes to the region of genomic DNA to which they aligned.  Overlaying genome-wide methylome and smRNA datasets confirmed increased methylation precisely within the stretch of DNA that matched the sequence of the smRNA.  Conversely, heavily methylated smRNA loci tended to spawn more smRNAs.

“We looked at a plant genome but our method can be applied to any system, including humans,” says Lister.  Although the human genome is about 20 times bigger than the genome of Arabidopsis – plant biologists’ favorite model system not least because of its compact genome – Ecker predicts that within a year or so, sequencing technology will have advanced far enough to put the 3 billion base pairs of the human genome and their methyl buddies within reach.

“This really is just the beginning of unmasking the role of these powerful epigenetic regulatory mechanisms in eukaryotes,” says Ecker.