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1.
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 DCsCl 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 DCsCl 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.  相似文献   

2.
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.  相似文献   

3.
Amyloid aggregation has been associated with numerous human pathological diseases. A recent study has demonstrated that silk fibroin intermittently endorses amyloidogenesis in vivo. In the current study, we explored the propensity of silk fibroin to undergo amyloid-like aggregation and its prevention using an optimized concoction of curcumin with β-cyclodextrin. Aggregation of silk fibroin resulted in the formation of fibrils with a diameter of ~3.2 nm. However, addition of the optimized concentration of curcumin and β-cyclodextrin to silk fibroin inhibited aggregation and preserved the random coil conformation even under aggregation inducing conditions, as demonstrated by CD and FTIR spectroscopy. Benzene rings of curcumin interact with the aromatic residues of fibroin via hydrophobic interactions. However, β-cyclodextrin preferentially interacts with the non-polar residues, which are the core components for nucleation dependent protein aggregation. The present study demonstrates the ability of the concoction of curcumin and β-cyclodextrin in tuning the self assembly process of fibroin. It also provides a platform to explore the assembly process of nano-fibril and hierarchical structures in vitro along with a novel insight for designing clinically relevant silk-based functional biomaterials.  相似文献   

4.
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.  相似文献   

5.
The fine structure of silk fibroin   总被引:1,自引:1,他引:0       下载免费PDF全文
The fine structure of Bombyx mori silk fibroin was investigated by electron microscopy and X-ray diffraction techniques. Examination of silk fibers fragmented with ultrasonic radiation and negatively stained revealed the presence of ribbon-like filaments of well-defined lateral dimensions. Analysis of the breadths of the equatorial reflections in the X-ray diffraction pattern of fibroin yielded similar dimensions for the lateral extent of the crystallites. It is concluded that the crystalline material in B. mori silk fibroin is in the form of ribbon-like filaments of considerable length parallel to the fiber axis and of lateral dimensions approximately 20 x 60 A.  相似文献   

6.
A procedure has been developed to obtain native fibroin in a pure state from the reservoir part of the silk gland. The purified protein has a sedimentation coefficient of 10 S as determined on sucrose density gradients and the amino acid composition is similar to that reported for fibroin from the cocoons. The effects of various solvents has been studied; lithium thiocyanate was found to be the solvent of choice. By in vivo labeling of fibroin with [3H]glycine and [14C]alanine it was demonstrated that fibroin synthesized in the posterior part of the gland and that stored in the reservoir part are identical.  相似文献   

7.
After in vivo labeling with [3H]glycine the synthesis and transport of fibroin has been studied by radioautography and cell fractionation.Radioactivity appearing in the cytoplasm is rapidly transferred to the lumen where it accumulates in the so-called silk layer before reaching the central core of secreted fibroin. By sucrose density gradients it was demonstrated that the radioactivity appears immediately in the fibroin fraction, no precursors being observed.A simple fractionation procedure, based on the utilization of detergent, gives three fractions tentatively interpreted as synthesis, transport, and accumulation compartments in accordance with their kinetics of labeling.  相似文献   

8.
A tissue-engineered mesh fabricated with adipose-derived mesenchymal stem cells (AD-MSCs) cultured on a silk fibroin scaffold is evaluated for use in female pelvic reconstruction. Thirty-five female Sprague Dawley rats were divided into four groups. Group A (n?=?10) were implanted with polypropylene meshes, Group B (n?=?10) with silk fibroin scaffolds and Group C (n?=?10) with tissue-engineered meshes. Group D (n?=?5) acted as the tissue control. The tissue-engineered mesh was produced as follows. AD-MSCs were obtained from adipose tissue of rats designated to Group C. The cells were seeded onto a silk fibroin scaffold, cultured and then observed by scanning electron microscopy (SEM). Histological studies of these meshes were performed at 4 and 12 weeks after implantation and mechanical testing was carried out on all groups before implantation and at 12 weeks after implantation. AD-MSCs displayed fibroblast-like shapes and were able to differentiate into adipocytes or fibroblasts. SEM observation showed that AD-MSCs proliferated and secreted a matrix onto the silk fibroin scaffolds. After implantation of the scaffolds into rats, histological analysis revealed better organized newly formed tissue in Group C than in controls. Group C also had a similar failure force (2.67?±?0.15 vs 2.33?±?0.38 N) and a higher Young’s modulus (2.99?±?0.19 vs 1.68?±?0.20 MPa) than a normal vaginal wall, indicating the potential of this tissue-engineered approach. AD-MSCs were validated as seed cells for tissue engineering. The silk fibroin scaffold thus shows promise for application with AD-MSCs in the fabrication of tissue-engineered mesh with good biocompatibility and appropriate mechanical properties for pelvic floor reconstruction.  相似文献   

9.
Silkworms contain a powerful and effective fibroin promoter, which controls the expression of fibroin, a silk protein. The fibroin promoter and well-known characteristics of silkworm, the application of transgenic technique to silkworm will provide an excellent opportunity to mass-produce biomolecules. In this study, the production of recombinant human insulin like growth factor-I (rhIGF-I) in the silkworm system was designed. The method makes use of the microinjection technique and P element vector to transfer foreign genes into the chromosomes. We constructed the expression vector using the fibroin gene promoter and P element vector containing IGF-I gene (pFpIGF-I). We then microinjected this vector into eggs, and through PCR screening, transgenic silkworms were selected. We isolated and purified rhIGF-I from silkworm cocoons, returning a concentration of rhIGF-I of about 1,300 ng/g from transgenic silkworm cocoons. In a comparison of transgenic silkworm rhIGF-I and colostral IGF-I on cell proliferation, colostral IGF-I was better able to increase the proliferation rate of the cell line relative to the transgenic silkworm rhIGF-I, and showed a similar cell proliferation pattern. The anti-cancer effects of transgenic silkworm rhIGF-I were higher than that of colostral IGF-I on HeLa and SNU-C1 cancer cells. These results confirmed the construction of new transgenic silkworm strains producing rhIGF-I.  相似文献   

10.
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.  相似文献   

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