Studies on silk fibroin of the silkworm Bombyx mori

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.     

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.     

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.     

The fine structure of silk fibroin

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.     

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.     

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.     

Genetic polymorphism of silk fibroin studied by two-dimensional translation pause fingerprints.

Silk fibroins from a number of B mori strains have been compared, and at least six different polypeptide size phenotypes, which show apparent molecular weights of 365,000–415,000 in SDS polyacrylamide gels, have been identified. Analysis of the corresponding fibroin messenger RNAs in denaturing gels shows that mRNA size is largely correlated with polypeptide length. The mRNAs vary in size from 5.50 × 10⁶ to 6.30 × 10⁶ daltons. It has been shown elsewhere that the translation of silk fibroin mRNA in a reticulocyte cell-free system proceeds discontinuously. In this paper, I demonstrate that this discontinuous translation phenomenon can be exploited to map the location of divergent amino acid sequences in fibroin variants. SDS gel analysis of translational pause patterns shows that divergence arises internally after a relatively long amino-terminal sequence which appears to be conserved. Two-dimensional gel analysis using V8 protease digestion in the second dimension produces fingerprints of fibroin peptide fragments ordered from the amino to the carboxyl terminus of the protein. These fingerprints provide additional evidence for extensive internal divergence of the fibroins and a reduced degree of divergence near the termini. A plausible explanation for the observed genetic variability is the occurrence of relatively large unequal crossing-over exchanges in the repetitive domain of the fibroin gene.     

Features of the cell-free translation of a spider fibroin mRNA

The massive production of fibroin by the large ampullate glands of the spider, Nephila clavipes, serves as a model system in which to study the synthesis and control of a large secretory protein. Their tissue-specific product, fibroin, produced during the entire adult life of the female, is approximately 320 kilodaltons, and rich in glycine and alanine. Highly purified fibroin mRNA from the glands has been translated in a rabbit reticulocyte cell-free system with variable supplements. The translational products analyzed by SDS-PAGE display two features, tRNA modulation and discontinuous pauses during elongation. tRNA complements exert their effects both in the translational efficiency and in the size of the peptides generated. The pauses observed during translation generate subsets of smaller discrete peptides, visualized in the gels as ladders of variable relative intensities, appearing exclusively and concomitantly with the fibroin. Definitive linkage between the discrete peptides and fibroin synthesis process has been established by their selective labeling with specific radioactive amino acids.     

The tritium labeling of small amounts of protein for analysis by electrophoresis on sodium dodecyl sulfate--polyacrylamide slab gels.

A simple and reproducible method for the tritium labeling of small amounts of proteins prior to analysis under denaturing conditions on polyacrylamide slab gels is described. The method involves the in vitro labeling of proteins by reductive methylation using formaldehyde and high specific activity [³H]potassium borohydride. Labeled proteins were detected by fluorography after fractionation on polyacrylamide slab gels in the presence of sodium dodecylsulfate. The overall procedure allows the analysis and molecular weight estimation of submicrogram quantities of protein.     

Photosynthetic Prokaryotes. Cell Differentiation and Functions : Edited by G.C. Papageorgiou and L. Packer Elsevier Biomedical; New York, 1983 405 pages. $65.00

A protein (designated as luffin) with an app. M_r of 26000, which inhibits protein synthesis in rabbit reticulocyte lysate, was purified to homogeneity from the seeds of Luffa cylindria roem by extraction with 20 mM Na phosphate buffer (pH 7.2) containing 0.2 M NaCl, ammonium sulfate fractionation, and chromatography on Sephacryl S-200 and CM-Sephadex C-25. Luffin exhibited 10-times as strong inhibitory activity against protein synthesis in rabbit reticulocyte lysate (IC₅₀, 0.42 ng/ml) as that of ricin A-chain, but it showed only a weak cytotoxicity against murine leukemia L1210 cells, an activity of 1/10⁶ to 1/10⁵ that of ricin.     

Identification of discrete electrophoretic components among the products of mitochondrial protein synthesis in HeLa cells.

Proteins synthesized in vivo by HeLa cell mitochondria in the presence of emetine, an inhibitor of cytoplasmic protein synthesis, have been characterized as to their electrophoretic mobility, solubility properties in organic solvents, and kinetics of labeling with [³H]isoleucine.Ten distinct electrophoretic components in the molecular weight range from about 11,000 to 42,000 have been identified with high reproducibility among the products of mitochondrial protein synthesis. Control experiments involving direct sodium dodecyl sulfate lysis of whole cells, or the use of the protease inhibitor phenylmethylsulfonylfluoride during cell homogenization and fractionation, have excluded a significant role of enzymic degradation during extraction in producing the observed electrophoretic pattern of mitochondrial protein products.A selective solubilization of mitochondrial protein products, representing a 20 to 30-fold purification with respect to the cytoplasmically synthesized proteins, has been obtained by using a neutral chloroform/methanol mixture. At least six of the electrophoretic components with a molecular weight greater than 20,000 were found to be extracted to an appreciable extent with neutral chloroform/methanol.A fairly co-ordinate labeling of the discrete electrophoretic components has been observed during a one-hour exposure period of the cells to [³H]isoleucine in the presence of emetine, without any clear evidence of intorconversion.     

Nitrogen Limitation in Natural Populations of Cyanobacteria (Spirulina and Oscillatoria spp.) and Its Effect on Macromolecular Synthesis

Natural populations of the cyanobacteria Spirulina species and Oscillatoria species obtained from Israeli fishponds were limited in growth by nitrogen availability in summer. Physiological indicators for nitrogen limitation, such as phycocyanin, chlorophyll a, and carbohydrate content, did not show clear evidence for nitrogen limited growth, since these organisms are capable of vertical migration from and to the nitrogen-rich bottom. By means of ¹⁴C labeling of the cells under simulated pond conditions followed by cell fractionation into macromolecular compounds, we found that carbohydrates synthesized at the lighted surface were partially utilized for dark protein synthesis at the bottom of these ponds.     

Quantitative analysis of photosynthetic carbon metabolism in protoplasts and intact leaves of barley. Determination of carbon fluxes and pool sizes of metabolites in different cellular compartments

Rates of carbon fluxes and pool sizes of photosynthetic metabolites in different cellular compartments of barley protoplasts were calculated from the time curves of their labeling in the medium of ¹⁴CO₂. Using membrane filtration procedure, kinetics of ¹⁴C incorporation into the products of steady-state photosynthesis was determined separately in chloroplasts, mitochondria and cytosol of barley protoplasts illuminated for different periods in the air containing ¹⁴CO₂. To extract the quantitative information, analytical labeling functions P(t) describing the dependence of ¹⁴C content in the primary, intermediate and end products of a linear reaction chain upon the duration of tracer feeding have been derived. The parameters of these functions represent pool sizes of metabolites and rates of carbon fluxes. The values of these parameters were determined by fitting the experimental labeling curves to the functions P(t) by means of non-linear regression procedure. To elucidate the possible effects of fractionation on the photosynthetic carbon metabolism, the parameters of protoplasts were compared with corresponding values in intact leaves of barley.     

In vitro production of Bombyx mori silk fibroin by organ culture of the posterior silk glands; isotope labeling and fluorination of the silk fibroin

An in vitro silk fibroin production system has been developed by culture of posterior silk glands from Bombyx mori. A large amount of the silk fibroin was produced continuously and effectively with a rotation culture procedure. Modified Grace's insect medium was used, and oxygen bubbling in the medium was performed. In addition, half of the medium was replaced with fresh medium every 6 h. The production yield of silk fibroin produced after 100 h culture was 81 mg/g wet weight of posterior silk gland. This culture system was used successfully for efficient (15)N isotope labeling of silk fibroin, which is required for (15)N solid state nuclear magnetic resonance (NMR) analysis of silk fibroin. Moreover, the introduction of fluorinated amino acids into silk fibroin was also carried out using this culture system. (c) 1993 John Wiley & Sons, Inc.     

Fractionation of the chymotryptic precipitate of Bombyx mori silk fibroin

1. A solution of Bombyx mori silk fibroin was digested with chymotrypsin. Amino acid analyses of the chymotryptic precipitate showed in addition to the main constituents Gly, Ala, Ser and Tyr, very small amounts of Lys, His, Arg, Asp, Thr, Glu, Pro, Cys, Val, Met, Ile, Leu, Phe and Trp. 2. A stable solution of the chymotryptic precipitate in 6m-urea was obtained by dialysing a solution in 50% (w/v) lithium thiocyanate against 6m-urea. 3. The dinitrophenylated chymotryptic precipitate in 6m-urea was fractionated by gel filtration and by ion-exchange chromatography. On Dowex 1 (X2), a main fraction I_d and three further fractions with different amino acid compositions and molecular weights were obtained. 4. Specific rearrangement and fission of the bonds involving the serine nitrogen atoms of fraction I_d and fractionation of the resulting mixture by gel filtration yielded five fractions. Two of these fractions had the compositions DNP-Ser-(Gly₆,Ala₄,Ser) and DNP-Ser-(Gly₄,Ala₂ or Ala₃,Ser) and are presumably double repeating units according to the proposed formula of Lucas, Shaw & Smith (1957), namely [Ser-Gly-(Ala-Gly)_n]₂, for n values of 2 and 1 respectively.