Abstract
RNA identified by its base composition and T1 RNase oligonucleotide pattern as the message for silk fibroin was purified from mature posterior silk glands of Bombyx mori larvae and used to direct polypeptide synthesis in an Ehrlich ascites cell-free extract. Fibroin mRNA stimulated [3-H]alanine incorporation about 3- to 4-fold in the presence of 80 mM K+ and 4 mM Mg-2+. The stimulation was reduced in the presence of 5 times 10-minus 6 to 10-minus 4 M aurintricarboxylic acid, an inhibitor of the initiation of protein synthesis. The cell-free products were heterogeneous in size, including peptides as large as 100,000 daltons. They co-precipitated with carrier fibroin sequences after digestion with trypsin. A large fraction of the polypeptides synthesized in response to fibroin mRNA was precipitated by antiserum directed against amino acid sequences in noncrystalline region polypeptides of fibroin. Furthermore, after digestion with chymotrypsin, a major fraction of the cell-free products specifically co-precipitated with crystalline region sequences of native fibroin. The size and amino acid composition of the fibroin crystalline region polypeptides isolated from the cell-free products were similar to those from native fibroin.
Highlights
More than 90% of the amino acids in fibroin are glycine, alanine, serine, and tyrosine, in a ratio of 44.5:29.4:12.1:5.2
The results indicate that cell-free polypeptides similar in amino acid composition to the authentic fibroin crystalline region polypeptides are precipitated by this technique (Table V)
The messenger RNA for silk fibroin was the first mRNA to be identified by chemical means
Summary
More than 90% of the amino acids in fibroin are glycine, alanine, serine, and tyrosine, in a ratio of 44.5:29.4:12.1:5.2. Chymotrypsin digestion of fibroin releases crystalline region polypeptides, which crystallize and precipitate. Crystalline region polypeptides contain 60% of the amino acids in the protein, and have a sequence proposed by Lucas et al [10] to be Gly-Ala-Gly-Ala-Gly[Ser-Gly-(Ala-Gly),l,-Ser-Gly-Ala-Ala-Gly-Tyr where n is usually 2 and has an average value of 2. The major noncrystalline region sequences remain soluble after chymotrypsin digestion of fibroin, yet except for the more frequent occurrence of tyrosine, resemble those sequences which precipitate. We have taken advantage of the unusual features of this protein to detect the cell-free synthesis of fibroin polypeptides by specific precipitation after digestion with trypsin and by a chymotrypsin digestion, co-crystallization technique which detects fibroin crystalline region polypeptides. An immune precipitation procedure which detects peptides in the noncrystalline region of fibroin has been used
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