COMPARATIVE STUDIES OF THE FIBROIN GENE OF VARIOUS SILK WORM SPECIES

Fibroin has been isolated from silk glands of several species of silk moths, Bombyx mori, Antheraea polyphemus and Samia cynthia. The large sizes of the various fibroin polypeptides (MW 220,000 to 350,000 daltons) indicate that their corresponding mRNA should contain a minimum coding sequence of 10,000 to 16,000 nucleotides.; Fibroin mRNAs isolated from the silk glands by sucrose gradient centrifugation were identified as fibroin-specific by the following criteria: (1) The mRNAs can only be detected in silk gland cells, where fibroin synthesis is most active. (2) They can be preferentially labeled with ('3)H-uridine, suggesting that these mRNA species are actively synthesized. (3) The isolated mRNa hybridize specifically to DNA with a G + C content expected for a fibroin gene and to bacterial plasmid DNA that contains silk fibroin gene of B. mori or S. cynthia. The concentration of fibroin gene sequences in S. cynthia DNA was measured by saturation hybridization of mRNA to total S. cynthia DNA. The saturation level, in conjunction with the genomic DNA content and the mRNA size indicated that there is only one fibroin gene per haploid S. cynthia genome.; Fibroin gene DNA of S. cynthia can be separated from bulk DNA by CsCl-Hoechst 33258 centrifugation. This partially purified DNA was used to construct recombinant plasmids. Upon screening with mRNA, several fibroin gene containing clones were obtained. They were characterized by restriction endonucleases cleavage. A. polyphemus silk fibroin gene containing clones were isolated by screening a bacteriophage library of A. polyphemus DNA. These clones were also characterized similarly.; The sequence divergence of the silk fibroin genes and total single-copy DNA of the three species was measured by thermal melting of the hybrids formed individual silk fibroin gene DNAs or single-copy DNAs. The result indicated that for closely related species, S. cynthia and A. polyphemus there is more sequence conservation for the fibroin gene (12%) than for the total single-copy DNA (17%). However, for the more distantly related B. mori and A. polyphemus or B. mori and S. cynthia, no specific conservation for structural gene sequences was observed.