University of Louisville

Poster Title

Systematic Analysis of a 7kda Vertebrate Pheromone From D. Ocoee

Institution

University of Louisville

Abstract

D. ocoee is a species of terrestrial salamander native to the mountainous regions of western North Carolina. This salamander uses pheromones during courtship to increase female receptivity. The male scratches the female’s skin with his premaxillary teeth to deliver the courtship pheromones from his submandibular mental gland to the female. The pheromone mixture contains sodefrin precursor-like factor, a 20 kDa protein, and other unidentified proteins. A related species P. shermani uses Plethodontid Modulating Factor (PMF), a 7-kDa protein related to snake cytotoxins and xenoxins, as a major component of its pheromone mixture. This pheromone is unusual in its highly conserved untranslated region (UTR). Over 50 different isoforms of PMF have been discovered in P. shermani. cDNA suggests that this protein may also be present in D. ocoee. Total RNA was extracted from the D. ocoee mental gland, amplified using specialized primers designed to anneal to the conserved UTR. The sequences were cloned, re-amplified, then sequenced, resulting in only one PMF sequence. This sequence matched the previously discovered PMF in D. ocoee 100%. Proteinaceous pheromones from D. ocoee were separated and characterized using size exclusion chromatography, strong anion exchange chromatography and reverse phase chromatography. Proteins collected were analyzed by ESI and MALDI-TOF analyses. Observed masses were in a range from 6090-20248 Da. Mass spectral data for one of the proteins matched a D. ocoee cDNA sequence with coverage of 64%.

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Systematic Analysis of a 7kda Vertebrate Pheromone From D. Ocoee

D. ocoee is a species of terrestrial salamander native to the mountainous regions of western North Carolina. This salamander uses pheromones during courtship to increase female receptivity. The male scratches the female’s skin with his premaxillary teeth to deliver the courtship pheromones from his submandibular mental gland to the female. The pheromone mixture contains sodefrin precursor-like factor, a 20 kDa protein, and other unidentified proteins. A related species P. shermani uses Plethodontid Modulating Factor (PMF), a 7-kDa protein related to snake cytotoxins and xenoxins, as a major component of its pheromone mixture. This pheromone is unusual in its highly conserved untranslated region (UTR). Over 50 different isoforms of PMF have been discovered in P. shermani. cDNA suggests that this protein may also be present in D. ocoee. Total RNA was extracted from the D. ocoee mental gland, amplified using specialized primers designed to anneal to the conserved UTR. The sequences were cloned, re-amplified, then sequenced, resulting in only one PMF sequence. This sequence matched the previously discovered PMF in D. ocoee 100%. Proteinaceous pheromones from D. ocoee were separated and characterized using size exclusion chromatography, strong anion exchange chromatography and reverse phase chromatography. Proteins collected were analyzed by ESI and MALDI-TOF analyses. Observed masses were in a range from 6090-20248 Da. Mass spectral data for one of the proteins matched a D. ocoee cDNA sequence with coverage of 64%.