Synthetic Chromosome

'Synthetic' Chromosome Permits Rapid, On-Demand 'Evolution' of Yeast; Artificial System Has Built-In Diversity Generator

In the quest to understand genomes -- how they're built, how they're organized and what makes them work -- a team of Johns Hopkins researchers has engineered from scratch a computer-designed yeast chromosome and incorporated into their creation a new system that lets scientists intentionally rearrange the yeast's genetic material.

A report of their work appears September 14 as an Advance Online Publication in the journal Nature.

"We have created a research tool that not only lets us learn more about yeast biology and genome biology, but also holds out the possibility of someday designing genomes for specific purposes, like making new vaccines or medications," says Jef D. Boeke, Ph.D., Sc.D., professor of molecular biology and genetics, and director of the High Throughput Biology Center at the Johns Hopkins University School of Medicine.

Boeke notes that yeast is probably the best-studied organism with a nucleus on the planet and is "already used for everything from medicine to biofuel," making it a good candidate for his team's focus.

In designing the synthetic yeast chromosome, Boeke says, the goal was to make it maximally useful to researchers by laying down some ground rules: First, the product could not compromise yeast survival; second, it must be as streamlined as possible; and third, it had to contain the capacity for genetic flexibility and change.

Using the already known full genetic code -- or DNA sequences -- of the yeast genome as a starting point, Johns Hopkins graduate student Sarah Richardson wrote a software program for making a series of systematic changes to the DNA sequence. The changes were planned to subtly change the code and remove some of the repetitive and less used regions of DNA between genes, and to generate a mutated "version 2.0" of a yeast cell's original 9R chromosome. The smallest chromosome arm in the yeast genome, 9R contains about 100,000 base pairs of DNA and represents about one percent of the single-celled organism's genome.

Building the actual chromosome started with stringing individual bases of DNA together that were then assembled into longer segments. Large segments of about 10,000 base pairs were finally put into live yeast cells and essentially swapped for the native counterpart in the chromosome, a process for which yeast are naturally adept. In addition to 9R, the team also made a smaller piece of the chromosome 6L. Yeast cells containing the synthetic chromosomes were tested for their ability to grow on different nutrients and in different conditions, and in each case came out indistinguishable from natural yeast.

The Hopkins teams says what distinguishes this constructed chromosome from the native version -- and sets it apart from other synthetic genome projects -- is an "inducible evolution system" called SCRaMbLE, short for Synthetic Chromosome Rearrangement and Modification by Lox-P mediated Evolution.

"We developed SCRaMbLE to enable us to pull a mutation trigger -- essentially causing the synthetic chromosome to rearrange itself and introducing changes similar to what might happen during evolution, but without the long wait," explains Boeke. Why build in the scrambling system? To change multiple things at once, says Boeke, which is anathema among experimental scientists who traditionally change only one variable at a time, Nature is never that well controlled, he says.

The team activated SCRaMbLE in yeast containing both the synthetic 9R and 6L chromosomes, then analyzed the DNA from the yeast cells. Testing this population of SCRaMbLEd yeast fed various nutrients they found some grew fast, some grew slowly and others really slowly, and some of the fast-growing ones had very specific defects resulting from specific gene loss, showing that SCRaMbLE does indeed introduce random variation. When the team analyzed the molecular structure of the synthetic 9R and 6L chromosomes from this SCRaMbLEd population, they found chromosomes with small deletions, rearrangements, and other alterations, at wildly varying locations.

"If you think of the yeast genome as a deck of cards, we now have a system by which we can shuffle it and/or remove different combinations of 5000 of those cards to get lots of different decks from the same starter deck," Boeke says. "While one derivative deck might yield good hands for poker, another might be better suited for pinochle. By shuffling the DNA according to our specifications, we hope to be able to custom design organisms that perhaps will grow better in adverse environments, or maybe make one percent more ethanol than native yeast."

Boeke says the 9R and 6L experiments are "the beginning of a big project, whose ultimate goal is to synthesize the whole yeast genome (about 6000 genes) and SCRaMbLE the 5000 likely to be individually dispensable. And he wants to make the tool available to anyone who wants to use it, without intellectual property protection.

Major support for this study came from the National Science Foundation, with other contributions from Microsoft, Department of Energy, and Fondation pour la Recherche Médicale.

In addition to Boeke, Johns Hopkins scientists who contributed to the Nature study are Jessica S. Dymond, Sarah M. Richardson, Candice E. Coombes, Timothy Babatz, Joy Wu Schwerzmann, Héloïse Müller, Narayana Annaluru, Annabel C. Boeke, Junbiao Dai, Srinivasan Chandrasegaran, and Joel S. Bader.

Also, William J. Blake of Codon Devices; and Derek L. Lindstrom, and Daniel E. Gottschling of Fred Hutchinson Cancer Research Center.

Blood Mystery Solved: Two New Blood Types Identified

Two New Blood Types Identified

You probably know your blood type: A, B, AB or O. You may even know if you're Rhesus positive or negative. But how about the Langereis blood type? Or the Junior blood type? Positive or negative? Most people have never even heard of these.

Yet this knowledge could be "a matter of life and death," says University of Vermont biologist Bryan Ballif.

While blood transfusion problems due to Langereis and Junior blood types are rare worldwide, several ethnic populations are at risk, Ballif notes. "More than 50,000 Japanese are thought to be Junior negative and may encounter blood transfusion problems or mother-fetus incompatibility," he writes.

But the molecular basis of these two blood types has remained a mystery -- until now.

In the February issue of Nature Genetics, Ballif and his colleagues report on their discovery of two proteins on red blood cells responsible for these lesser-known blood types.

Ballif identified the two molecules as specialized transport proteins named ABCB6 and ABCG2.

"Only 30 proteins have previously been identified as responsible for a basic blood type," Ballif notes, "but the count now reaches 32."

The last new blood group proteins to be discovered were nearly a decade ago, Ballif says, "so it's pretty remarkable to have two identified this year."

Both of the newly identified proteins are also associated with anticancer drug resistance, so the findings may also have implications for improved treatment of breast and other cancers.

As part of the international effort, Ballif, assistant professor in the biology department, used a mass spectrometer at UVM funded by the Vermont Genetics Network. With this machine, he analyzed proteins purified by his longtime collaborator, Lionel Arnaud at the French National Institute for Blood Transfusion in Paris, France.

Ballif and Arnaud, in turn, relied on antibodies to Langereis and Junior blood antigens developed by Yoshihiko Tani at the Japanese Red Cross Osaka Blood Center and Toru Miyasaki at the Japanese Red Cross Hokkaido Blood Center.

After the protein identification in Vermont, the work returned to France. There Arnaud and his team conducted cellular and genetic tests confirming that these proteins were responsible for the Langereis and Junior blood types. "He was able to test the gene sequence," Ballif says, "and, sure enough, we found mutations in this particular gene for all the people in our sample who have these problems."

Transfusion Troubles

Beyond the ABO blood type and the Rhesus (Rh) blood type, the International Blood Transfusion Society recognizes twenty-eight additional blood types with names like Duffy, Kidd, Diego and Lutheran. But Langereis and Junior have not been on this list. Although the antigens for the Junior and Langereis (or Lan) blood types were identified decades ago in pregnant women having difficulties carrying babies with incompatible blood types, the genetic basis of these antigens has been unknown until now.

Therefore, "very few people learn if they are Langereis or Junior positive or negative," Ballif says.

"Transfusion support of individuals with an anti-Lan antibody is highly challenging," the research team wrote in Nature Genetics, "partly because of the scarcity of compatible blood donors but mainly because of the lack of reliable reagents for blood screening." And Junior-negative blood donors are extremely rare too. That may soon change.

With the findings from this new research, health care professionals will now be able to more rapidly and confidently screen for these novel blood group proteins, Ballif wrote in a recent news article. "This will leave them better prepared to have blood ready when blood transfusions or other tissue donations are required," he notes.

"Now that we know these proteins, it will become a routine test," he says.

A better match

This science may be especially important to organ transplant patients. "As we get better and better at transplants, we do everything we can to make a good match," Ballif says. But sometimes a tissue or organ transplant, that looked like a good match, doesn't work -- and the donated tissue is rejected, which can lead to many problems or death.

"We don't always know why there is rejection," Ballif says, "but it may have to do with these proteins."

The rejection of donated tissue or blood is caused by the way the immune system distinguishes self from not-self. "If our own blood cells don't have these proteins, they're not familiar to our immune system," Ballif says, so the new blood doesn't "look like self" to the complex cellular defenses of the immune system. "They'll develop antibodies against it," Ballif says, and try to kill off the perceived invaders. In short, the body starts to attack itself.

"Then you may be out of luck," says Ballif, who notes that in addition to certain Japanese populations, European Gypsies are also at higher risk for not carrying the Langereis and Junior blood type proteins.

"There are people in the United States who have these challenges too," he says, "but it's more rare."

Other Proteins

Ballif and his international colleagues are not done with their search. "We're following up on more unknown blood types," he says. "There are probably on the order of 10 to 15 more of these unknown blood type systems -- where we know there is a problem but we don't know what the protein is that is causing the problem."

Although these other blood systems are very rare, "if you're that one individual, and you need a transfusion," Ballif says, "there's nothing more important for you to know."