LONDON The strange observation that some vertebrate species use one method to generate diverse immunoglobulin molecules while others use another may not seem so strange after all, now that researchers in Germany have discovered that the same gene controls both methods.
Jean-Marie Buerstedde, director of the Institute of Molecular Radiobiology in Munich, and his colleagues reported in the Feb. 15, 2002, Science that the gene that encodes the enzyme known as activation-induced deaminase (AID) controls all of the processes specific to B cells that generate diversity in the immunoglobulin genes of these cells.
The work is reported in a paper titled “Requirement of the Activation-Induced Deaminase (AID) Gene for Immunoglobulin Gene Conversion.”
Buerstedde, who was at the Heinrich Pette Institute in Hamburg, Germany, when he carried out the work for the Science paper, is keen to establish partnerships with biotechnology companies to develop his findings further. His next target is to try to identify how AID exerts its action. “We want to know whether it directly modifies DNA or whether it activates another gene, which then regulates repertoire development,” he told BioWorld International.
The body needs to produce many B cells, each of which manufactures a different immunoglobulin molecule or antibody, so that there is always one that is capable of making antibodies that will bind to whatever foreign protein enters the body. How this was possible was a source of puzzlement to generations of immunologists. Scientists initially had calculated that there might not even be enough room in the genome for all the genes that would be needed to encode such a large variety of immunoglobulin molecules.
Then researchers discovered that the genes encoding the immunoglobulin molecules in B cells are combined from families called V, D and J segments. This is known as site-specific V(D)J recombination and it results in what is called the primary repertoire. Given the variety of infectious agents, however, this process by itself does not provide enough variety.
A process called somatic hypermutation introduces a further level of diversity, by inducing point mutations into the rearranged genes. Some of these mutations inactivate the immunoglobulin genes, but others provide the chance to make an antibody molecule that is an even better fit for an antigen that the B cell may one day encounter.
When scientists began to study various organisms, however, they realized that not all vertebrates use the same methods to generate their repertoires of B cells. Chickens and rabbits, they found, use a process called gene conversion, which involves sequence exchanges between different V segments. Sheep, by contrast, use only somatic hypermutation, but humans and mice use both V(D)J recombination and somatic hypermutation to develop their antibody diversity.
Buerstedde said, “This seemed very strange. It was as though chickens and rabbits were sitting on one branch of the evolutionary tree and sheep, mice and humans on another. This made us believe that somatic hypermutation and gene conversion were somehow related.”
His latest experiments show that the missing link is AID. He and his colleagues used a cell line from chickens called DT40, which is derived from B cells, and which undergoes B-cell gene conversion in cell culture.
The team showed several years ago that if they transfected genes into this cell line, the DNA becomes integrated at very high frequency into the equivalent endogenous gene. Buerstedde speculated that this might be because chicken B cells need homologous recombination for immunoglobulin gene conversion.
“So to test the function of the AID gene, we did something similar to a mouse knockout experiment, but in these cells,” Buerstedde said, “we took a construct containing a defective AID gene and added it to the cells. We then looked at the gene conversion activity of these mutant cells and found that gene conversion had stopped. When we transfected cDNA containing the AID gene into the mutant cells, gene conversion began again.
“This is a very exciting finding because it shows that apart from the process known as V(D)J recombination, all phenomena that contribute to the development of a diverse repertoire of immunoglobulin molecules are regulated in the same way,” he said. “In summary, the two methods of developing a repertoire, known as somatic hypermutation and gene conversion, are dependent on the AID gene.”
Buerstedde also said the DT40 cell line has great potential as a means of testing the function of various genes. “I think people will now start to investigate the function of many genes using this cell line because it is easier and quicker to produce the mutant clones than doing knockout mouse experiments. The only caveat is that one has to be able to measure the mutant phenotype in cell culture.”