August 13, 1999
Single Switch Triggers Two Immune System Genes
Two genes critical to the immune system's adaptability in battling
viruses, bacteria and other invaders receive on and off signals from a
single DNA segment, HHMI researchers have found. The discovery explains
how the two genes work in concert and hints at how the genes have
managed to remain partners for more than 450 million years since they
first appeared in cartilagenous fish as part of an adaptive immune
system.
In an article in the August 13, 1999, issue of the journal
Science, HHMI investigator Michel
Nussenzweig and colleagues at The Rockefeller University reported
that neighboring genes, called RAG1 and RAG2, are
switched on in concert by a single genetic control signal nestled near
RAG2 on the chromosome. The discovery is like finding that a
light switch in one house also controls lights in a house across the
street.
“The big question is how do you regulate something that lands in the genome in such a way that both of these gene products, which are required for the transposase activity, in fact are coordinately regulated.”
Michel C. Nussenzweig
The RAG1 and RAG2 genes produce proteins that somehow
join to form a "transposase," an enzyme that helps snip apart and
rearrange genes that code for two critical weapons in the immune
system's arsenal. These weapons are protein antibodies produced by the
immune system's B cells and receptors found on the surface of T
cells.
Antibodies are molecules that roam the bloodstream, recognizing and
attaching to invaders and marking them for destruction. Similarly, the
receptors on T cells—the immune system's infection—fighting
"soldiers"—are those that recognize antigens, fragments of foreign
proteins, so thatthe T cells can attack the viral or bacterial
proteins.
The RAG1/RAG2 recombinases' ability to rearrange the genes for such
antibodies and receptors is crucial to the immune system's flexibility
in generating a nearly infinite variety of weapons against infections
and malignant cells. HHMI investigator David
Schatz discovered RAG1 and RAG2 in 1989, and in 1998 with
colleagues at Yale University, he showed that RAG1 and RAG2 had
transposase activity.
"Before our studies, we knew that the two genes were controlled
coordinately," said Nussenzweig. "However, we knew very little about
their control elements." He added that previous experiments testing
small segments of DNA were inconclusive in defining the
RAG1/RAG2 control mechanism.
In the current work, however, Nussenzweig and his colleagues used
specially modified bacterial artificial chromosomes (BACs) to carry
large pieces of DNA into mouse cells. HHMI investigator Nathaniel
Heintz, also at Rockefeller, invented the BAC modification
technique.
Using BAC to shuttle DNA into the mice, the researchers created
transgenic mice that possessed fluorescently-tagged RAG genes
and extended DNA segments adjacent to the RAG genes that the
researchers suspected carried the RAG controller. In addition,
Nussenzweig's team used the BACs to introduce genes coding for two
fluorescent proteins-one that would shine green wherever RAG1 appeared,
the other yellow in cells expressing RAG2.
By inserting such test segments into mice and looking for
fluorescent B and T cells, the scientists could determine whether those
segments contained a control sequence that properly switched on the RAG
genes.
"We used the marker genes to show us where the RAGs were being
expressed, and then we used recombination technology to knock off
pieces of the bacterial artificial chromosomes to reveal where the
control elements might be," explained Nussenzweig.
After many such experiments with various constructed segments, the
scientists narrowed down the control sequence to a small piece of DNA
next to one end of the RAG2 gene.
The discovery that the neighboring RAG genes are controlled
by a single switch has important implications for understanding how the
two genes evolved as part of the immune system, said Nussenzweig.
"The configuration of these genes makes scientists believe that they
were inserted into the genome together, as a 'transposable element,'"
he said. What's more, said Nussenzweig, the finding may explain why,
over the 450 million years since the genes first appeared, evolution
has required them to remain closely spaced in the genome.
"The big question is how do you regulate something that lands in the
genome in such a way that both of these gene products, which are
required for the transposase activity, in fact are coordinately
regulated?"
The key to the genes' long-lived marriage might lie in the mechanism
by which the control sequence activates both genes at once, said
Nussenzweig. In the Science paper, he and his colleagues propose
two possibilities: First, the control element may bounce back and forth
between the promoter regions of the two genes and activate them;
alternatively, the controller may touch both simultaneously.
Although Nussenzweig emphasizes that there are no data that reveal
which is the correct mechanism, the theory of simultaneous activation
might better explain why evolution has refused to allow the two genes
to separate and still remain functional partners in the transposase
enzyme. He and his colleagues plan experiments that they hope will
reveal the control mechanism for RAG1 and RAG2.
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