The Case of the Missing Gene
By Randy Weckman
hen you think about it, it's not such a big intellectual leap from studying genes in a fungus that infects rice and certain other grass family members to investigating
mutations that cause birth defects in babies. You need only to understand that genes work pretty much the same way in all organisms, including fungi, humans, cats, dogs, and donkeys, among others.
That's why the March of Dimes organization was interested in funding the research of Mark Farman in the Department of Plant Pathology. That research is on mutations in a fungus that causes a disease in rice called rice blast. Rice and fungi wouldn't seem at first blush to be in the March of Dimes field of interest, but Farman's shining of a big, bright light on the process of gene loss in the fungus, the March of Dimes believed, might well help medical researchers understand the same phenomenon that causes all manner of birth defects in babies. And that's a worthwhile goal as far as the March of Dimes is concerned, because preventing birth defects has been its raison d'etre since it was established in the 1930s.
Plant Pathologis Helps March of Dimes with Research
The research of Farman, a northwest-Londoner by birth, is at the below-tiny, below-microscopic level; he studies the lilliputian world of DNA (genetic material) of the rice blast fungus Magnaporthe grisea. This rice fungus is important to world rice production because it can waste a rice crop posthaste. And the fungus also can cause severe devastation in certain susceptible turf grasses such as annual ryegrass, perennial ryegrass, and St. Augustine grass, to name a few.
||Mark Farman (left), associate professor in Plant Pathology,
and Yun-Sik Kim, a post-doctoral scholar, have been researching
gene loss in a particular rice fungus. The March of Dimes
funded the project, which may eventually lead to better
understanding of how gene loss plays a part in birth defects.
But this fungus has a peculiarity that makes Farman take an interest in it. Its quirk is that when one particular strain of the fungus is crossed with certain other strains, a gene that controls pigment in the fungus is lost in about 25 percent of the progeny. Because that gene causes color, progeny without the gene are pale and wan, easily recognized. Sometimes, however, when strains that are known to be genetically unstable are crossed with other strains, there is no gene loss. Weird, huh?
Furthermore, those 25 percent that lack pigmentation can't manufacture an enzyme (tetrahydroxynaphthalene reductase) that allows the fungus to synthesize its own melanin.
Normally, melanin allows the fungus to be stiff enough to penetrate rice leaves, enabling it to establish an infection. Because 25 percent of the strains don't have the melanin mechanism that allows infection to occur, the genetic mutant seems to be self-limiting. Progeny with the gene loss can no longer cause disease; nonetheless, the mutation appears with regularity as a new (de novo, as geneticists call it) defect.
And while 25 percent may seem an almost magical number to those who've had an elementary course in Mendelian genetics (Aha! A simple recessive trait will be expressed when two recessive genes come together after fertilization, and that will occur in 25 percent of progeny), as a fungal geneticist, Farman knew that the phenomenon he saw and recorded was truly due to gene loss. That's because the rice blast fungus has only one set of chromosomes, so the recessive trait would be expressed 100 percent of the time.
Farman compared the genetic material of the mutants with parent fungi and found that the mutants, plain and simple, had a big hole where a gene that codes for melanin synthesis should be. It had been deleted sometime during meiosis. Meiosis is the process by which a cell's double set of chromosomes becomes a single set in the sperm or egg. Offspring from the fertilized egg then receive one set of chromosomes from each parent. See the sidebar on meiosis on page 15 if you need a quick refresher course in biology. If the oddity was caused instead by the expression of a non-functional gene, once reproduction occurred there would be a copy of that gene.
The Human Connection
Why is chromosome loss more than a novelty, a sideshow for a fungus that causes disease in rice? Does this phenomenon have implications for science beyond allowing researchers to understand and maybe control a disease in rice and certain turf grasses?
De novo gene lossesthose in which the gene loss occurs in the first generation and have not been inheritedalso are relatively common in plants and animals. Often, the results are catastrophic. Wolf-Hirschhorn syndrome in humans, for example, is caused by the deletion of genes on the human chromosome 4; the syndrome is manifested in growth and mental retardation, incomplete brain development, and other defects. Several congenital heart defects are caused by genetic aberrations on chromosome 22. The Angelman syndromesometimes called the happy puppet syndrome because children born with it have severe mental retardation, inappropriate laughter, lack of speech, and herky, jerky gaitis thought to be caused by genetic errors in the 15th chromosome.
Because Farmans rice fungus is rather predictable (at least in the sense that genes are deleted with high frequency), it is a good organism to study to find out exactly whyand howgene deletion occurs. If Farman's research in the rice fungus, which is truly basic science, can answer these puzzling questions, other researchers may be able to discover ways to prevent a variety of birth defects in babies.
Farman has shown that fungal strains possessing an unstable pigment gene have small pieces of DNA on each side of the gene that are virtually identical. Such repeats can cause problems for the fungal cells, especially if they are close to one another. This is because the enzymes that replicate the chromosomes can jump between repeats, failing to copy any of the DNA in between. During normal meiosis, these jumps are prevented because a matching chromosome from the other parent pairs tightly with the unstable chromosome, holding the repeats well apart.
Farman proposes that the frequent de novo mutations occur when the chromosome region containing the pigment gene is organized differently in each of the parents. This prevents the partners that are pairing from aligning correctly, which will allow the repeats surrounding the unstable pigment gene to pair with each other. If the cell then performs what it believes to be a normal meiotic exchange of DNA strands in this self-paired loop, the pigment gene will be released from the chromosome and degraded.
Real-World Problem Solving
|But a simple association does not a theory prove.
That's why researchers always work from a theoretical rationale, which seeks to explain how two observations are related. Researchers develop their rationale and then try to disprove it through crucial research.
In a series of experiments, Farman made the crosses between the unstable strain and stable strains and found that pigment gene deletions occurred whenever chromosome pairing was predicted to be disrupted.
Furthermore, deletions occurred only in the chromosome that possessed the repeats on each side of the gene; the sites of deletion were right within the repeated gene, Farman said. Even more telling, however, was the finding that the unstable chromosome region rarely suffered deletion in crosses where it was able to pair with an identical chromosome, he said.
Farman thus was able to specify that the pigment deficiency is caused by gene loss and was able to predict with precision the circumstances under which losses occurred when parents have chromosomes with different organizational patterns in a region rich in repeated DNA.
How does this help manage the disease in rice and turf grasses?
According to plant pathologist Paul Vincelli, who collaborated with Farman on discovering a strain of Magnaporthe grisea fungus that causes devastating disease on perennial ryegrass, many fungicides lose their punch after a short time because the fungus adapts genetically so that it can surviveand sometimes thrivein the presence of the fungicide.
Understanding the basic molecular aspects of genetic variation in various fungal strains can help other scientists develop control agents that will remain effective against a constantly mutating fungal population, Vincelli said.
Aerial portion of the fungal structure in which meiosis occurs (top and center photos).
On the tip of the tool on the left are five to
six perithecia (cylinder-shaped sexual structures)
of the fungus being studied in Farmans lab.
One of them will be dissected and analyzed to see
if it suffered a genetic mutation.
Did the March of Dimes Get Its Moneys Worth?
Because the genetics of de novo chromosomal abnormalities isn't easy to study in humans, Farman's research on a fungus may provide important information on understanding how mutations occur. They appear not to be random events, but rather have a very strong, deterministic genetic basis. If scientists can understand the mechanism for such mutations, it may be possible to predict when such birth defects might occur, perhaps even develop methods to avoid them altogether if there is a strong probability of their occurrence.
Yun-Sik Kim, a post-doctoral scholar who has worked on the March of Dimes project.
In meiosis, a cells double set of chromosomes becomes a single set in the sperm or egg, so that after fertilization occurs, the egg has one set of chromosomes from each parent. Heres how it works (each chromosome as drawn is representative of a set of chromosomes):
||1. Most cells in our bodies contain two copies of each chromosome, one that came from Mom (blue) and the other from Dad (red).
2. Just before meiosis, chromosomes copy themselves, resulting in sister strands of DNA joined at the centromere.
3. Early in meiosis, each replicated chromosome pairs up with its partner in the middle of the cell so that the four DNA strands are aligned with one another. Enzymes cut the DNA strands at random points along the chromosomes. Another set of enzymes then joins the free ends to one of the strands on the partner chromosome, resulting in a crossover. In normal meiosis, the crossovers occur at exactly the same point on the two DNA strands, so no genetic information is lost.
4. After crossing over is complete, the chromosome pairs are pulled apart by cellular motors that latch onto their centromeres.
5. Finally, the sister strands are separated, resulting in four chromosome sets that are each packaged into a single sperm or egg cell, which are called haploid cells because they contain just one copy of each chromosome.
||Farman proposes that frequent de novo mutations occur when the chromosome region containing the pigment gene doesnt align correctly with its pairing partner during meiosis. This would allow repeats (of DNA) surrounding the unstable pigment locus to pair with each other.
The ABCs of Meiosis
If you think Watson refers to Sherlock Holmes sidekick and Crick refers to the pain in the neck you have from reading Sir Arthur Conan Doyle's classics about Sherlocks adventures, you might need a tutorial on meiosispro-nounced my - oh - sis. Meiosis occurs when reproductive cells (eggs or sperm) are produced. Suffice it to say, these days biology is more than carving up really dead animals.
Watson and Crick were the young scientists at Cambridge University in England (Watson was American; Crick was English) who in 1953 described that the genetic molecule (DNA) is made up of two chains of chemicals (sugar phosphates) arranged around a central axis much like a ladderexcept that the ladder is twisted into what's called a double helix. The ladderand each species has a certain number of discrete ladders in every cellis made up of chromosomes. (Humans have 46 chromosomes; goldfish have 94; gorillas, 48; and cattle, 60).
Continuing the ladder analogy, think of the rungs. Each rung is formed from the weak bonding of two of four basic molecules-adenine, cytosine, thymine, and guanine. Up and down the ladder are several rungs that in a row make up genes. And between rungs (genes)and this is important when understanding Farman's workare pieces of DNA that apparently have little importance but occupy spaces between genes. It is in these spaces (repeats) that Farman believes trouble begins.
De Novo Chromosomal Abnormalities
De novo chromosomal aberrations in humans are frequent. In fact, all mutationseven if they are inheritedstarted out as de novo aberrations. Syndromes in humans that are thought to be the outcome of chromosomal errors include some well-known ones, such as Down syndrome and dwarfism. Down syndrome occurs in one out of every 1,250 births to mothers in their 20s and in one in 30 births for women who are in their mid-40s. It is associated with a chromosomal defect on the 21st chromosome. Down syndrome (like several other birth defects such as Patau syndrome, Turner syndrome, and Kleinfelter syndrome) is caused by an incomplete separation of chromosome pairs during meiosis. Each of the syndromes shows three chromosomes (trisomy) when there normally would be only two. Further, many of the de novo chromosomal abnormalities are unnoticed because the fetus dies before it is detected as a pregnancy.
And while many genetic abnormalities are catastrophic, some are a real benefit, especially in agriculture.
Genetic aberrations in plants can lead to quite convenient foods. Seedless grapes and watermelon are two common foods that are the result of errors during meiosis that lead to the progeny having three sets of chromosomes instead of two. In the trade such plants are called triploid. The fact that no seeds are produced means that all new plants must be propagated using unconventional techniques. In grapes, new plants are created by coaxing cuttings from a mother plant to set roots and begin anew. In the case of watermelon, seeded varieties are chemically treated so that the resulting plants have four sets of chromosomes. Seeds from those plants are then crossed with a conventional two-set watermelon, which results in the triploid progeny that produces seedless fruit when pollinated with a two-sets-of-chromosomes variety.