Abstract
A physical map of the DNA containing the gene for silk fibroin was developed from direct hybridization analysis of restriction endonuclease digests of total Bombyx mori DNA using fibroin 125I-mRNA. The orientation of mRNA transcription relative to this map was deduced from the sensitivity of the mRNA coding strand within certain DNA restriction segments to lambda-exonuclease and exonuclease III. The map includes the entire gene coding region (Mr approximately 11 x 10(6)) and large DNA elements which flank the gene at its 5' end (Mr approximately 3 x 10(6)) and 3' end (Mr approximately 6.5 x 10(6)). The coding region is remarkably uniform in its sensitivity to restriction endonucleases. It is completely devoid of sites for most of the enzymes tested, including Hae III, the recognition sequence (d-pG-G C-C) of which might be expected to occur frequently in this large DNA block of 60% G + C content. The fibroin coding region does contain an enormous number of sites for enzymes predicted to have activity from known fibroin mRNA sequences. These results suggeste that the fibroin gene core is a large homogeneously repetitive block of DNA with little evidence for sequence divergence, or the presence of qualitatively different sequences, which might creat other restriction sensitivities. The map also allowed a comparison to be made of the fibroin gene "context" in DNA from tissues either active or inactive in fibroin synthesis.
Highlights
A physical map of the DNA containing the gene for silk fibroin was developed from direct hybridization analysis of restriction endonuclease digests of total Bombyx mori DNA using fibroin ‘““I-mRNA
Mapping of Sites for EcoRI, Barn I, and HindIII -The silk fibroin gene sequence has been identified in restriction endonuclease digests of total Born& mori DNA by specific hybridization with purified fibroin mRNA labeled with ““I. DNA
We have developed a physical map of the DNA surrounding the gene for silk fibroin in the genome of the silkworm B. mori
Summary
Electrophoresis, and Hybridization of DNA -Experiments were performed with B. mori DNA isolated from the middle silk gland, except where otherwise noted (Gage and Manning, 1976). Sal I in 6 mM Tris/HCl, pH 7.9, 6 rnM MgCl,, 6 rnM 2-mercaptoethanol, and 0.1 M NaCl. Multiple enzyme digests were usually carried out simultaneously in buffer specified for one of the enzymes. In cases where 2-mercaptoethanol and/or gelatin is required for activity, the buffer for this enzyme is used in the multiple digests. The DNA in the gel was denatured, transferred, and immobilized onto a nitrocellulose filter by the method of Southern (197513). The. DNA on the filter was hybridized overnight (16 to 18 h) with 5 to 7 rig/ml of fibroin ‘““I-mRNA and nonspecific radioactivity was removed under conditions ureviouslv published DNA which had been digested with EcoRI, otherwise the DNA bound actinomycin D poorly and did not band well during buoyant density centrifugation. A-Exonuclease was generously supplied by Dr John Chase, Albert Einstein Medical College and used according to Little (1967); 0.25 unit was used/fig of DNA
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