Production of a chimeric fibroin light-chain polypeptide in a fibroin secretion-deficient naked pupa mutant of the silkworm Bombyx mori.
The allelic Nd-s and Nd-sD mutations of the silkworm Bombyx mori are mapped to the same locus as that of the fibroin light (L)-chain gene (Fib-L). The silkworm carrying the homozygous Nd-s or Nd-sD mutation secretes less than 0.3% of the normal level of fibroin and produces a thin cocoon (naked pupa). In this study, cDNA sequences of the Nd-s and Nd-sD L-chains were compared with the cDNA and genomic sequences of the L-chain of the B. mori J-139 strain, a normal-level producer of fibroin. The two mutant cDNA sequences are almost identical except for one base change in the coding region. The N-terminal half of the L-chain encoded by exons I to III is identical between the mutants and J-139, but the rest of the molecule is completely different. The C-terminal half of the Nd-sD mutant L-chain is encoded by two exons, IV' and V', which are brought into proximity with the exon III by recombination between sequences in the third intron and in the far downstream region with concomitant loss of a region containing exons IV to VII. Sequences corresponding to exons IV' and V' are present about 10 kb downstream of the L-chain gene in the J-139 genome. Their homologous sequences have not been found in the DNA and protein databases. The chimeric L-chain molecule of about 27 kDa is present in posterior silk glands of Nd-s and Nd-sD strains without disulfide-bonding to the fibroin heavy (H-) chain, as revealed by Western blotting with the antibody specific to the C-terminal half of the mutant L-chain.
Mori
K
K
Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, Sendai, Japan.
Tanaka
K
K
Kikuchi
Y
Y
Waga
M
M
Waga
S
S
Mizuno
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D012831:Bombyx / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D005091:Exons; D005349:Fibroins / Q000096:biosynthesis* / Q000737:chemistry; D007438:Introns; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011679:Pupa / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D004220:Disulfides; D011993:Recombinant Fusion Proteins; D005349:Fibroins