The genes for silk fibroin in Bombyx mori

The genes for the protein silk fibroin were quantitated by hybridizaton of purified fibroin messenger RNA with the DNA from several tissues of the silk-worm Bombyx mori.     

Localization of the genes for silk fibroin in silk gland cells of Bombyx mori.

Radioactive iodinated silk fibroin messenger RNA and ribosomal RNA have been used as probes to localize their genes in tissue sections of Bombyx mori by in situ hybridization. From filter hybridization experiments it is inferred that the majority of the grains produced by in situ hybridization with fibroin mRNA represents specific hybridization to fibroin genes. Sections of the posterior silk gland where silk is synthesized have been compared with those of the middle gland which does not synthesize fibroin. Glands have been analyzed from the second through the fifth (last) larval instar during feeding and moulting periods. During later stages when the gland cells increase their DNA content by polyploidization, serial sections were required to follow the distribution of grains through entire nuclei. At all stages, both ribosomal DNA and fibroin genes are distributed randomly throughout the nuclei without a preferred relationship to any nuclear structure.     

Determination of the multiplicity of the silk fibroin gene and detection of fibroin gene-related DNA in the genome of Bombyx mori.

The number of silk fibroin genes per genome in the silkworm Bombyx mori has been determined by hybridization using fibroin [¹²⁵I]mRNA. The purified [¹²⁵I]mRNA had an oligonucleotide pattern after RNAase T₁ digestion which was characteristic of fibroin mRNA (Suzuki &; Brown, 1972) and it hybridized specifically to DNA with a G + C content expected for a fibroin gene. Thermal denaturations indicated that these hybrids were mismatched by about 3%, which probably indicates some variation among the sequences encoding the internal repetitions of the fibroin protein.The concentration of fibroin gene sequences in B. mori DNA was measured by saturation hybridization of [¹²⁵I]mRNA to filter bound DNA. The same saturation level of 1.8 × 10^?5 μg mRNA per μg DNA was calculated from data obtained with unfractionated DNA and with fibroin gene sequences which had been separated from bulk B. mori DNA by actinomycin $\text{D}\text{CsCl}$ centrifugation. Scatchard plots of the subsaturation data extrapolated to an identical saturation value. Internal reiteration of the fibroin mRNA molecule was apparent from the high association constant of hybridization. An exhaustive hybridization experiment showed that such repetitions comprise at least 90% of each mRNA molecule. The saturation value, in conjunction with the genome DNA content and the mRNA size, indicated the presence of only one fibroin gene per haploid B. mori genome.Hybridization of actinomycin $\text{D}\text{CsCl}$ fractionated DNA indicated that fibroin mRNA can form hybrids with DNA that bands with bulk B. mori DNA. These hybrids appear to involve DNA which is related to, but distinguishable from, true fibroin gene sequences. The fibroin gene-related sequences form mismatched hybrids with the mRNA, are much shorter than the fibroin gene and are dispersed in B. mori DNA of much lower G + C content, and there are many copies of these sequences per B. mori genome.     

Beta structure of periodic copolypeptides of L-alanine and glycine. Their relevance to the structure of silks

The β structure of seven periodic copolypeptides of l-alanine and glycine² namely: A₂G, AG, A₂G₂, A₃G₃, A₂G₃, AG₂ and AG₃ has been studied by infrared, X-ray and electron diffraction techniques. The sheets are made of anti-parallel chains and intersheet spacing is observed to depend both on residue sequence and composition. Samples with glycine molar fractions varying from 0.33 to 0.6 are found to be good models for group II of silk fibroins. The structure of Bombyx mori silk fibroin is discussed in the light of these new data.     

Expression of the Japanese oak silkworm Antheraea yamamai fibroin gene in the domesticated silkworm Bombyx mori

Abstract To understand the evolutionary conservation of the gene expression mechanism and secretion machinery between Antheraea and Bombyx fibroins, we introduced the genomic A. yamamai fibroin gene into the domesticated silkworm, B. mori. The spliced A. yamamai fibroin mRNA appeared only in the posterior region of the silk gland of the transgenic silkworm, suggesting that the functions of the fibroin promoter region and the splicing machinery are conserved between these two species. The A. yamamai fibroin protein was detected in the lumen of the silk gland of the transgenic silkworm, albeit at lower levels compared with the B. mori‐type fibroin. We found a strong degeneration of the posterior region of the silk gland of the transgenic silkworm. As a result, the cocoon shell weight was much lower in the transgenic silkworm than in the non‐transgenic line. These results indicate that the promoter function and splicing machinery are well conserved between A. yamamai and B. mori but that the secretion mechanism of fibroin is diversified between the two.     

Isolation and identification of the messenger RNA for silk fibroin from Bombyx mori

The messenger RNA for the protein silk fibroin has been isolated from the posterior silk gland of Bombyx mori and identified by partial sequence analysis. The sequence of mRNA could be predicted because the protein has a simple repetitious primary structure in which glycine residues comprise 45% of all residues and alternate predominantly with alanine and serine.     

Quantitative measurements of fibroin messenger RNA synthesis in the posterior silk gland of normal and mutant Bombyx mori

The amount of newly synthesized and accumulated fibroin messenger RNA has been measured quantitatively at various stages of posterior silk gland development in Bombyx mori. The two-step method involves fractionation on a Bio-Gel column which excludes the large mRNA, followed by RNAase T₁ digestion, and fractionation of the oligonucleotides on DEAE-Sephadex. Larvae in the feeding stages of the third and fourth instar synthesize and accumulate fibroin mRNA to about 2% of cellular RNA; this corresponds to 0.2 and 2 μg per pair of posterior glands in the third and fourth instars, respectively. More than 70% of this mRNA is degraded in vivo during the third and fourth moulting stages. Fibroin mRNA synthesis resumes again within the first 24 hours of the fifth instar; the mRNA accumulates and predominates over other DNA-like RNAs as the stage proceeds until finally it comprises about 3.5% of cellular RNA in a mature larva (170 μg per pair of posterior glands). These results indicate that more than 99% of the fibroin mRNA detected in the fifth instar is synthesized during this stage.Four spontaneous mutants of B. mori which synthesize very low levels of fibroin have been analyzed for their RNA content in the middle fifth instar. The total cellular RNA of the posterior gland is reduced to 4 to 7% of normal. Fibroin mRNA is more severely reduced to 1% of normal. In three heterozygotes, which have mutant phenotypes with respect to fibroin production, only slight increases of total cellular RNA and fibroin mRNA were observed. Thus, the primary biochemical lesion in these mutants is still unknown.The presumed ancestor to B. mori, the wild silkworm B. mandarina, was also analyzed for its fibroin mRNA. The mRNA isolated from fifth instar larvae of B. mandarina is indistinguishable from that of B. mori with respect to its nucleotide sequence, molecular weight and fraction of total cellular RNA.     

固定化过氧化物酶丝素膜的制备及其性质

家蚕丝素经高浓度的中性盐氯化钙溶解后,制成了固定化过氧化物酶丝素膜,对这种酶膜的活性和理化特性作了分析,结果表明这种酶膜的活性高,酶促反应温度范围宽,最适pH5.0-7.0,热稳定性也较游离酶好,这与用溴化锂溶解丝素后制成的固定化过氧化物酶膜相仿.因此,用这种方法制成的丝素膜同样是一种良好的固定化酶的生物材料.     

LIM-homeodomain transcription factor Awh is a key component activating all three fibroin genes,fibH, fibL and fhx,in the silk gland of the silkworm,Bombyx mori

In the silkworm Bombyx mori, three fibroin genes, fibroin-heavy-chain (fibH), fibroin-light-chain (fibL) and fibrohexamerin (fhx), are coexpressed only in the posterior silk gland (PSG) cells, while the sericin genes encoding silk glue proteins are expressed in the middle silk gland (MSG) cells. Silk gland factor-2 (SGF-2) is a PSG-specific activator complex of fibH, composed of a LIM-homeodomain protein, Awh, and its cofactors, Ldb and Lcaf. We investigated whether SGF-2 can activate other fibroin genes using transgenic silkworms. The genes for Ldb and Lcaf were expressed ubiquitously in various tissues, while the gene for Awh was expressed strictly specific in PSG of the wild type silkworms. Misexpression of Awh in transgenic silkworms induced ectopic expression of fibL and fhx as well as fibH in MSG. Coincidently with the induction of fibL and fhx by Awh, binding of SGF-2 to the promoter of fibL and fhx was detected in vitro, and SGF-2 binds directly to the fhx core promoter. Ectopic expression of the fibroin genes was observed at high levels in the middle part of MSG. Moreover, fibL and fhx were induced in the anterior silk gland (ASG) of the transgenic silkworms, but fibH was not. These results indicate that Awh is a key activator of all three fibroin genes, and the activity is probably regulated in conjunction with additional factors.     

Silk fibroin model,poly(l-alanylglycyl-l-alanylglycyl-l-alanylglycyl-l-seryglycine)

l-Alanylglycyl-l-alanylglycyl-l-alanylglycyl-l-serylglycine and its pentachlorophenyl ester methanesulphonate have been synthesized as monomers for the preparation of silk fibroin model polypeptide. The former octapeptide was polymerized with diphenylphosphorylazide (DPPA) and triethylamine in DMSO or in HMPA—pyridine, and the latter octapeptide pentachlorophenylester was polymerized by adding triethylamine in DMSO to give poly(l-alanylglycyl-l-alanylglycyl-l-alanylglycyl-l-serylglycine). This sequential polypeptide gave a similar i.r. pattern to the crystalline part of Bombyx mori silk fibroin, which indicated antiparallel β-conformation. Dialysis of the solution of this polymer in 60%, aqueous LiBr against water gave mainly the polymer of α-form. O.r.d. measurements suggest that this polypeptide exists as a random structure in dichloroacetic acid on in 60% aqueous LiBr.     

Silk—A new substrate for UDP-d-xylose: Proteoglycan core protein β-d-xylosyltransferase

The formation of most connective tissue polysaccharides is initiated by transfer of d-xylose from UDP-d-xylose to specific serine residues in the core proteins of the putative proteoglycans. The substrate specificity of the xylosyltransferase catalyzing this reaction has not yet been examined in detail, but it appears that a -Ser-Gly- pair is an essential part of the substrate structure. Since the preparation of the known acceptors (e.g., Smith-degraded or HF-treated cartilage proteoglycan) involves a substantial effort, we have searched for readily available proteins with the -Ser-Gly-sequence, which might serve as alternative substrates. In the present work, it was found that silk fibroin from Bombyx mori, which consists, in large part, of the repeating hexapeptide, Ser-Gly-Ala-Gly-Ala-Gly, is an excellent substrate for the xylosyltransferase from embryonic chick cartilage. Pieces of silk were used directly in the reaction mixtures, and [¹⁴C]xylose transferred from UDP-d-[¹⁴C]xylose was measured by liquid scintillation spectrometry after rinsing the silk in 1 m NaCl and water. Substantially greater incorporation was observed with preparations of silk or fibroin which had been dissolved in 60% LiSCN and subsequently dialyzed exhaustively or diluted appropriately. Under standard reaction conditions, the V_max for fibroin was 531 pmol/h/mg enzyme protein, as compared to 223 pmol/h/mg for Smith-degraded proteoglycan. K_m values were 182 mg/liter (fibroin) and 143 mg/liter (Smith-degraded proteoglycan). The product of [¹⁴C]xylose transfer to silk was alkali labile, and [¹⁴C]xylitol was formed when [¹⁴C]xylosylsilk was treated with borohydride in alkali. Proteolytic digestion with papain, Pronase, leucine aminopeptidase, and carboxypeptidase A yielded a radioactive product which was identified as [¹⁴C]xylosylserine by electrophoresis and chromatography. The identity of the isolated [¹⁴C]xylosylserine was further supported by its resistance to treatment with alkali (0.5 m KOH: 100°C; 8h) and by acid hydrolysis which yielded [¹⁴C]xylose. Tryptic and chymotryptic fragments from fibroin were also good xylose acceptors and had V_max values 60–70% of that observed for the intact protein. Substantial acceptor activity was displayed also by the sericin fraction of silk and by the silk sequence hexapeptide, Ser-Gly-Ala-Gly-Ala-Gly; the latter had a V_max value close to 20% of that of intact fibroin.     

Ultrastructure of fibroin in the silk gland of larval Bombyx mori

The fibroin molecules stored in Golgi vacuoles in the posterior silk gland cells of 72-h-old, fifth instar larvae of Bombyx mori L. were observed electron-microscopically. The fibers which float in the Golgi vacuoles often have their ends attached to the limiting membrane. The fibers are helical bundles about 130 Å in diameter composed of 5–7 threads, each 20–30 Å thick.     

YAC介导的天蚕丝素基因向家蚕的转移 ——天蚕丝素基因—YAC克隆在家蚕的表达

本文为天蚕Antheraea yamamai丝素基因在家蚕Bombyx mori成功表达的首次报道。     

Osteochondral Tissue Engineering In Vivo: A Comparative Study Using Layered Silk Fibroin Scaffolds from Mulberry and Nonmulberry Silkworms

The ability to treat osteochondral defects is a major clinical need. Existing polymer systems cannot address the simultaneous requirements of regenerating bone and cartilage tissues together. The challenge still lies on how to improve the integration of newly formed tissue with the surrounding tissues and the cartilage-bone interface. This study investigated the potential use of different silk fibroin scaffolds: mulberry (Bombyx mori) and non-mulberry (Antheraea mylitta) for osteochondral regeneration in vitro and in vivo. After 4 to 8 weeks of in vitro culture in chondro- or osteo-inductive media, non-mulberry constructs pre-seeded with human bone marrow stromal cells exhibited prominent areas of the neo tissue containing chondrocyte-like cells, whereas mulberry constructs pre-seeded with human bone marrow stromal cells formed bone-like nodules. In vivo investigation demonstrated neo-osteochondral tissue formed on cell-free multi-layer silk scaffolds absorbed with transforming growth factor beta 3 or recombinant human bone morphogenetic protein-2. Good bio-integration was observed between native and neo-tissue within the osteochondrol defect in patellar grooves of Wistar rats. The in vivo neo-matrix formed comprised of a mixture of collagen and glycosaminoglycans except in mulberry silk without growth factors, where a predominantly collagenous matrix was observed. Immunohistochemical assay showed stronger staining of type I and type II collagen in the constructs of mulberry and non-mulberry scaffolds with growth factors. The study opens up a new avenue of using inter-species silk fibroin blended or multi-layered scaffolds of a combination of mulberry and non-mulberry origin for the regeneration of osteochondral defects.