Date of Award
Program or Major
Doctor of Philosophy
Edward K Tillinghast
The orb web of Argiope aurantia was partitioned into a water-soluble and insoluble fraction. The water-soluble fraction was composed of low molecular weight (LMW) molecules ($<$5 kDa) and middle molecular weight (MMW) molecules (14.3 kDa-29 kDa). Choline was one of the principal components of the LMW molecules. This study demonstrated that spiders, like many insects, are unable to synthesize choline from glucose. Attempts to sequence one of the purified MMW proteins were unsuccessful due to the blockage of the N-terminus.
The major emphasis of this research centered on the major ampullate silk of Argiope aurantia. Attempts were made to characterize this silk, both by direct amino acid sequencing and inference from the sequence of a cDNA library clone for one of the major ampullate silk proteins.
To isolate the progenitive silk from the lumen of the major ampullate gland, it was first necessary to inhibit the spider proteases which are known to cleave silk. No commercially available proteinase inhibitors proved effective. Spider hemolymph was then observed to contain highly effective proteinase inhibitors. These inhibitors were observed in the hemolymph of all orb-weavers studied. Typically, 10 volumes of hemolymph were required to neutralize one volume of spider digestive fluid. Progenitive silk could be cleaved by vertebrate trypsin to produce water-soluble and insoluble fragments. Neither fraction was amenable to further analysis.
The total RNA from the major ampullate gland was isolated and a cDNA library generated. A clone for what is probably one of two major ampullate proteins was isolated and sequenced. This sequence contains a 1611 base coding region that consists of 1290 bases coding for a series of repeating amino acids, 321 bases coding for a nonrepetitive region and an 84 base noncoding region. The repetitive region is similar to the repetitive region of Nephila clavipes Spidroin 1. The nonrepetitive region has areas similar to both Nephila clavipes Spidroin 1 and 2. This partial sequence for Argiope aurantia also demonstrates a codon bias for codons with adenine or thymine in the third position.
Tugmon, Cathy Rose, "The characterization of the orb web of Argiope aurantia (Araneae: Araneidae) with emphasis on the major ampullate silk" (1993). Doctoral Dissertations. 1750.