Isolation of Lactodectus Hespersus spider silk genes.
Poster Number
13
Format
Poster Presentation
Abstract/Artist Statement
The two major proteins that make up dragline silk, major ampullate spidroin 1 (MaSpl) and 2 (MaSp2), have been cloned in different species of spider. To search for the homologues in L. hesperus (the black widow), we searched the literature and found conserved regions in the amino acid sequence of the MaSpl and MaSp2 proteins across species, and assumed that those same motifs would be conserved in black widow silk. We made degenerate primers and obtained small fragments of cDNA corresponding to these regions; we used these retrieved cDNAs as probes to screen a silk gland cDNA library for longer gene pieces. We have isolated approximately 4 kb cDNA fragments of MaSpl and MaSp2. We have partially sequenced each cDNA. A similar approach is being used to isolate the gene that comprises egg case silk from black widow spiders. This approach is a slight modification of the steps mentioned above due to the fact that there is no sequence information available from other species. For isolation of egg case cDNA, we have obtained peptide fragments from purified egg case silk and determined their amino acid sequence using matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. Designing primers from these fragments, we have performed PCR and isolated the gene.
Location
Pacific Geosciences Center
Start Date
26-4-2003 9:00 AM
End Date
26-4-2003 5:00 PM
Isolation of Lactodectus Hespersus spider silk genes.
Pacific Geosciences Center
The two major proteins that make up dragline silk, major ampullate spidroin 1 (MaSpl) and 2 (MaSp2), have been cloned in different species of spider. To search for the homologues in L. hesperus (the black widow), we searched the literature and found conserved regions in the amino acid sequence of the MaSpl and MaSp2 proteins across species, and assumed that those same motifs would be conserved in black widow silk. We made degenerate primers and obtained small fragments of cDNA corresponding to these regions; we used these retrieved cDNAs as probes to screen a silk gland cDNA library for longer gene pieces. We have isolated approximately 4 kb cDNA fragments of MaSpl and MaSp2. We have partially sequenced each cDNA. A similar approach is being used to isolate the gene that comprises egg case silk from black widow spiders. This approach is a slight modification of the steps mentioned above due to the fact that there is no sequence information available from other species. For isolation of egg case cDNA, we have obtained peptide fragments from purified egg case silk and determined their amino acid sequence using matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. Designing primers from these fragments, we have performed PCR and isolated the gene.