Genetic polymorphism of silk fibroin studied by two-dimensional translation pause fingerprints

Abstract

Silk fibroins from a number of B mori strains have been compared, and at least six different polypeptide size phenotypes, which show apparent molecular weights of 365,000–415,000 in SDS polyacrylamide gels, have been identified. Analysis of the corresponding fibroin messenger RNAs in denaturing gels shows that mRNA size is largely correlated with polypeptide length. The mRNAs vary in size from 5.50 × 10 6 to 6.30 × 10 6 daltons. It has been shown elsewhere that the translation of silk fibroin mRNA in a reticulocyte cell-free system proceeds discontinuously. In this paper, I demonstrate that this discontinuous translation phenomenon can be exploited to map the location of divergent amino acid sequences in fibroin variants. SDS gel analysis of translational pause patterns shows that divergence arises internally after a relatively long amino-terminal sequence which appears to be conserved. Two-dimensional gel analysis using V8 protease digestion in the second dimension produces fingerprints of fibroin peptide fragments ordered from the amino to the carboxyl terminus of the protein. These fingerprints provide additional evidence for extensive internal divergence of the fibroins and a reduced degree of divergence near the termini. A plausible explanation for the observed genetic variability is the occurrence of relatively large unequal crossing-over exchanges in the repetitive domain of the fibroin gene.