FEATURE ARTICLE
Tracking Down a Cheating Gene
Some genes will play dirty to gain a selective advantage
Barry Ganetzky
Distorters
At the same time that other laboratories were learning about Responder, people in my lab were trying to understand the other half of the problem. We were trying to make sense of Sd—the key gene required for distortion. This was difficult, since we had no idea what the gene looked like and didn't know whether we would recognize it if we did in fact come across it.
On the basis of its genetic properties, we anticipated that the mutant gene—Sd—would differ from its normal counterpart—Sd+—by more than just a single nucleotide base. We believed the difference to be more substantial, and therefore that it could be readily discerned by standard molecular biological methods. Using cytogenetic techniques, John B. Brittnacher extended my original deletional analysis and identified a chromosome segment of roughly 200,000 bases in which Sd was located. Patricia Powers cloned this entire region as a series of small overlapping fragments. She then compared each fragment from the SD chromosome, which carries the mutant gene, with the corresponding fragment from normal chromosomes.
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The comparison revealed only a single difference. A particular fragment was about 6,500 nucleotide bases long on the normal chromosome but was almost twice as long—12,000 bases—on the SD chromosome. Further analysis revealed the reason for this size difference. A segment of DNA is duplicated on the mutant chromosome.
Since this was the only detectable difference between the normal and the distorting chromosome, we concluded that it contained at least part of the Sd gene. But we needed to know whether it contained all of Sd.
To find out, Janna McLean and Cynthia Merrill injected the DNA fragment we believed to contain the distorter gene into Drosophila embryos containing only normal chromosomes. The inserted genes can become incorporated in the DNA of some of these embryos. We predicted that if we actually had the Sd gene, that embryos receiving and integrating the inserted DNA would acquire the ability to produce offspring that could cause distortion. This in fact is what happened.
Now that we knew that our inserted DNA fragment contained a gene or genes capable of inducing full distorting ability, we hoped we could identify the Sd gene itself and determine its function.
From her analysis, Merrill determined that the normal fragment of 6,500 bases actually contains two overlapping genes. One of these is the Drosophila counterpart of a mammalian gene encoding heparan-sulfate-2-sulfotransferase (HS2ST). The second gene encoded the Drosophila counterpart of a protein known in yeast and mammals as RanGAP. RanGAP has recently been shown to be an essential component of a complex system that transports proteins and RNA molecules into and out of the cell's nucleus.
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We discovered that both the HS2ST and the RanGAP genes are represented twice on the SD chromosome, as opposed to just once on the normal counterpart. Both genes appear to be normal on the right hand portion of the duplication, as does the HS2ST gene on the left. But the RanGAP gene on the left is not normal; rather it encodes a mutant RanGAP that lacks the last 234 amino acids.
Because this truncated RanGAP protein was the only substantially altered protein encoded in the SD fragment, we concluded that this was the one responsible for the distorting activity. If that were true, we expected to be able to find the truncated protein in the testes of distorting males. Leyla Bayraktaroglu and Ayumi Kusano demonstrated that the truncated protein is indeed found in the testes of distorting males, as is the normal-sized protein. In contrast, normal males produce only the normal-size protein.
To obtain decisive proof that we had the right gene, we once again injected a DNA fragment into normal embryos to create distorting flies. This time, Merrill inserted only the left half of the fragment—the one that contains the truncated RanGAP gene. (Since this gene overlaps with the HS2ST gene, it is impossible to insert RanGAP alone. Both genes were inserted together, but HS2ST was disabled and rendered nonfunctional. Only the truncated RanGAP protein could be produced from this fragment.)
The results from this experiment were unequivocal. Flies that received the engineered DNA fragment acquired the ability to cause distortion with the same strength as flies carrying a native SD chromosome. Therefore, we concluded that the truncated RanGAP is indeed the functional Sd product.
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