July 22, 2004 A paper describing discoveries about the role of a gene that is important in all animals, plants, and fungi is published in the 20 July 2004 issue of the journal Proceedings of the National Academy of Sciences. One of the discoveries is that the gene, named RAD51, plays an essential role in the the process of recombining the genetic material in chromosomes during sexual reproduction in plants. In humans, defects in this process can cause a fetus to have abnormal numbers of chromosomes, resulting in infertility, miscarriages, or birth defects. The new discoveries about the gene's role in plants suggest that it also may have an essential role in the production of sperm and egg cells in humans and other mammals.
One of the most surprising results of the research is that the RAD51 gene is not essential for survival in plants, as it is known to be in mammals. "It is well known that a mouse fetus that inherits two defective copies of the RAD51 gene will die very soon after conception, so we were quite surprised to find that our mutant plants, which have two defective copies of this gene, develop quite normally--except that they are sterile," reports Hong Ma, professor of biology at Penn State University and the leader of the research team that made the discoveries in collaboration with the Bernd Reiss group at the Max-Planck Institute for Plant Breeding Research at Cologne, Germany.
The team's work reveals specific functions of the protein products of the RAD51 gene, including the pairing of compatible chromosomes--the structures that contain the cell's genes--during the process of meiosis, when an organism's reproductive cells form during its development, and also the repairing of breaks in the chromosomes that occur during this and other process. "Our research leads us to suspect that plant cells repair breaks in their DNA in a different way from mammal cells, which just stop growing if the RAD51 gene is not functioning," Ma says.
Meiosis produces the cells of sexual reproduction, such as sperm cells and egg cells, which contain only a single strand of each of the organism's characteristic number of chromosomes. Later, when the mature sperm and egg cells combine, the new individual will have its full complement of DNA--half from the mother and half from the father. Much of the team's research is focused on the reshuffling of genetic material between two similar chromosomes, which occurs during meiosis when the double-stranded chromosomes in the cell's nucleus are pulled apart and brought together again twice by a team of proteins, resulting in four new male or female reproductive cells.
The researchers conduct their studies with a mutant strain of the model laboratory plant, Arabidopsis, in which the RAD51 gene is unable to function because a piece of foreign DNA is inserted in the middle of the gene. Ma's team dissected the plant's tiny flower buds before they had a chance to develop, when they were only about 0.3 or 0.4 millimeters in diameter. "It is important to look at the cells from the tiny flower buds before they open because meiosis is an event that occurs before pollen development starts," Ma explains. "We then treat the cells with chemicals that protect their structure from damage and then stain them with a chemical that allows us to see the DNA much more clearly than any other part of the cell.
The researchers' microscopic images of meiosis in their mutant plants revealed not neat chromosome packages but a chaos of many broken sections of chromosomes. "We found that plants in which the RAD51 gene does not function are not able to recombine the sections of their chromosomes that are broken during meiosis," Ma says. The researchers additionally tested this finding by introducing into the plants a mutation in a gene, named SPO-11, that disables the protein system that cuts chromosomes. "In plants that are defective in both SPO-11 and RAD51, we find intact chromosomes, not the jumble of chromosome fragments," Ma reports. "Because the chromosomes are not cut in the first place, you don't really need the RAD51 to repair it." These experiments demonstrate that RAD51 has an essential role in the biochemical repair of DNA during recombination. They also establish that this system for repairing broken chromosomes, which previously was known to occur in animals and fungi, occurs in plants, as well.
The RAD51 gene codes for a type of enzyme known as a recombinase, which catalyzes the swapping, or "crossover," of DNA sections between very similar, or "homologous" chromosomes. At conception, an individual inherits one copy of a chromosome from its father and another very similar but not exactly identical copy from its mother. This DNA swapping--a process known as recombination--is important for generating diversity among individuals within the same family and throughout the entire the population.
These very similar molecules of DNA, along which specific genes lie at the same point on each strand, are brought together by the molecular machinery of the cell to form a two-chromosome structure known as a homologous pair. Because the DNA along each chromosome is so similar, a thin structure is able to form between the chromosomes resulting in a structure known as a synaptonemal complex, which links the two strands together. Ma's research is the first to provide definitive evidence that the RAD51 gene is required specifically for homolog pairing and synapsis, in addition to its role in recombination during meiosis. "In the mutant, we found that the chromosomes do not come together to form pairs," he reports. "Our research shows that the RAD51 protein has a role in bringing a single strand of DNA to its counterpart within the nucleus of a cell to form a double-strand and is a critical component of the whole complex of proteins that holds the two strands close to one another."
Many of the findings of the Ma's team's research are revealed is its exquisite electron-microscope images, which show more clearly than ever before the process of cell division in plants. "We are fortunate to have on our team a scientist who is highly skilled at the very tricky process of preparing cells for viewing with an electron microscope," Ma says. Like a loaf of bread cut into slices, the images show a section-by-section dissection of the chromosomes that reveal their structure and position within the cell. Clearly visible is the jelly-sandwich-like structure known as the synaptonemal complex--a thick piece of the chromosome sitting next to another thick piece with a thin central element joining the two. This synaptonemal complex should form during meiosis for the entire set of chromosomes in normal plant cells, but Ma found that it does not form in the reproductive cells of the mutant plant. "You just see lots of thick pieces that have not gotten paired with another piece because RAD51 is not there to bring the two pieces together," Ma says. The electron-microscope images show definitively that the RAD51 gene is required in plants for forming the central element that pairs the two chromosomes.
The RAD51 gene also is known to be required for its role in repairing damage caused to DNA in all of an organism's cells by harmful radiation and chemicals. The cell uses an intact section of DNA as a template for repairing a damaged homologous one. The research establishes that this system for repairing broken chromosomes with the protein products of the RAD51 gene, which previously was known to occur in animals and fungi, occurs in meiotic cells of plants, as well.
"Our research suggests that some defects in human reproduction may be associated with partial loss of the RAD51 function," Ma says. "While it is not possible at present to study defects in this RAD51 gene in people who have fertility problems, we now can use this mutant plant strain to understand more than we could before about its role in sexual reproduction."
In addition to Ma and Reiss, other members of the research team include Wuxing Li and Changbin Chen at Penn State; Ullrich Markmann-Mulisch and Elmon Schmelzer at the Max-Planck Institute in Germany; and Ljudmilla Timefejeva at Penn State and the Estonian Agricultural University in Estonia. This research was supported by grants from the U. S. National Institutes of Health, the U.S. National Science Foundation, the U. S. Department of Energy, and the European Commission.
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