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At least 19 recordsLinked to original sources
PubMed1980

Internal structure of the silk fibroin gene of Bombyx mori. I The fibroin gene consists of a homogeneous alternating array of repetitious crystalline and amorphous coding sequences.

The DNA sequence orgainzation of the protein encoding region of the gene for silk fibroin has been analyzed. The accompanying paper (Manningm R. F., and Gage, L. P. (1980) J. Biol. Chem. 255, 9451-9457) shows that the total length of the gene, and its protein, as well as the pattern of restriction sites in the gene is highly polymorphic among inbred stocks of Bombyx mori, In this paper, those features of fibroin gene structure which are invariant among these alleles are presented. Fibroin is composed primarily of relatively short "crystalline" and "amorphous" peptides of known sequence whose arrangement in the protein is unknown. Knowledge of the codons most commonly used in fibroin mRNA allowed utilization of particular restriction inzymes as a means for determing the nature and organization of crystalline and amorphous coding sequences in the fibroin gene. Three restriction endonucleases were identified that cleve sequences coding for amorphous region peptides. Their cleavage pattern revelaed that the repetitive coding sequence of the gene core (approximately 15 kilobases) is divided into at least 10 large crystalline coding domains interrupted by smaller amorphous coding domains. Many restriction endoncleases do not cleave the fibroin core at all, three of them with four gase recognition sequences. Specific deductions as to codon usage and repetitive sequence homogeneity in the gene follow from these results. One novel finding is the rigorous exclusion of the glycine codon GGA prior to serine codons even though this glycine codon is used frequently prior to alanine codons. The sequence homogeneity and the regularly alternating arrangement of crystalline and amorphous coding sequences of the gene are discussed in terms of the function of fibroin protein and the evolution of highly repetitive DNA.

Alleles↗
PubMed1984

Natural fibroin genes purified without using cloning procedures from fibroin-producing and -nonproducing tissues reveal indistinguishable structure and function.

Natural fibroin genes were purified from total DNA extracted from the fibroin-producer cells (posterior silk gland) and -nonproducer cells (middle silk gland or pupa) by two cycles of CsCl/actinomycin D centrifugation followed by sucrose density gradient centrifugation. Purity of the final samples was greater than 14%. DNA sequences of these natural genes between positions -171 and +104 were identical and showed no sign of base modification as assayed by the method of Maxam and Gilbert. The determined sequence includes the promoter and a major part of the modulator. When assayed in an in vitro transcription system prepared from middle silk gland, template activities of the purified natural fibroin genes from the producer and the nonproducer were indistinguishable from that of cloned fibroin DNA. Digestion and blotting of total genomic DNAs with several restriction enzymes that recognize methylation changes on DNA revealed no difference of hybridization pattern of fibroin DNAs in a region from -650 to +326 between the producer and nonproducer. Thus, it is unlikely that the differential transcription of the fibroin gene is controlled by a change of base modification in the regions of transcription signals.

Animals↗
PubMed1991

Purification and characterization of an enhancer-binding protein of the fibroin gene. II. Functional analyses of fibroin factor 1.

Fibroin factor 1 (FF1) when coupled with fibroin factor 2 (FF2) is an enhancer binding protein of the fibroin gene. FF1a, one type of FF1, has been purified to homogeneity from crude nuclear extracts of posterior silk gland cells and identified as a protein with molecular mass 125 kDa (Suzuki, T., Matsuno, K., Takiya, S., Ohno, K., Ueno, K., and Suzuki, Y. (1991) J. Biol. Chem. 266, 16935-16941). FF1 is able to recognize and bind a sequence of -205 to -185 in the enhancer I of the fibroin gene in the presence of FF2. The binding sequence of FF1 with FF2 contains two repeats of the derivative of consensus sequence which is recognized by homeobox-containing proteins. Though FF1 activity to construct the major band complex I is specific to posterior silk gland cells, use of a specific antibody raised against FF1a showed that FF1 protein is ubiquitous in Bombyx cells. These results suggest the possibility that FF1 molecules present as multiple proteins might be specifically modified and activated for the binding to enhancer DNA in posterior silk gland cells. Since the FF1a antibody also inhibited a transcriptional enhancement activity governed by the enhancer sequence, we conclude that FF1 is one of the transcriptional factors of the fibroin gene. The functions of FF1 on fibroin gene transcription are discussed.

Antibodies↗
PubMed1976

Studies on silk fibroin of Bombyx mori. I. Fractionation of fibroin prepared from the posterior silk gland.

1. Fractionation of fibroin prepared from the posterior silk glands of Bombyx mori was carried out. After carboxymethylation of the fibroin, it was fractionated by ammonium sulfate precipitation, Sephadex G-200 gel filtration and DEAE-cellulose column chromatography. 2. The fibroin was composed of at least two protein groups of large molecular size and three or four components of small molecular size, and, in addition, a mixture of proteins ranging in size from about 25,000 to more than 100,000 daltons with almost the same amino acid compositions. 3. The latter proteins contained about 48% glycine, 32% alanine, 11% serine, 4.5% tyrosine, 2% valine, and other minor amino acids. The sum of these main five amino acids accounts for more than 97% of the total amino acid residues of the proteins. 4. The present results indicate major heterogeneity in the molecular size of posterior silk gland fibroin, and, in addition, suggest the possibility of repeating sequences with relatively simple amino acid compositions in major peptide chains of fibroin.

Amino Acids↗
PubMed1980

Isolation of giant silk fibroin polysomes and fibroin mRNP particles using a novel ribonuclease inhibitor, hydroxystilbamidine.

Hydroxystilbamidine isethionate, a dye capable of binding to both DNA and RNA, has been found to be a powerful inhibitor of cellular ribonucleases. A procedure has been developed that, with the aid of this compound, permits the preparative isolation of giant silk fibroin polyribosomes from the posterior silk gland of Bombyx mori. The polyribosomes contain approximately 45-112 ribosomal particles, as judged by electron microscopy. Treatment of giant fibroin polyribosomes with EDTA releases a particle that sediments at 125S. This mRNP particle contains biologically active silk fibroin mRNA, as judged by cell-free translation in an mRNA-dependent reticulocyte cell-free system.

Bombyx↗
PubMed1991

Purification and characterization of an enhancer-binding protein of the fibroin gene. I. Complete purification of fibroin factor 1.

An enhancer-binding protein of the fibroin gene, fibroin factor 1 (FF1), has been purified to homogeneity from crude nuclear extracts of posterior silk gland cells where this gene is transcribed specifically. There is a multiplicity of FF1; the FF1 activity was eluted as at least three major fractions on column chromatographies. FF1 is able to form a stable complex with the enhancer DNA sequence in the presence of another proteinous factor named FF2, which lacks ability to bind DNA molecules by itself. One of FF1 forms, FF1a, was purified with a combination of classical purification techniques without using a sequence-specific affinity column, and identified as a protein with molecular mass 125 kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To obtain homogeneous protein of FF1a, purification of more than 26,000-fold from the starting nuclear extract was necessary.

Animals↗
PubMedDate not supplied

Development stage-specific expression of fibroin in the silk worm Bombyx mori is regulated translationally.

The contents of fibroin H RNA as a function of development have been quantitated in the posterior silk glands of Bombyx mori larvae on different days of 4th and 5th instars. The fibroin RNA levels increased during the feeding stages of larvae and the RNA got completely degraded during the interim moult. The patterns of accumulation of fibroin RNA were similar in both the instars. Although there was considerable increase in the fibroin RNA content during the 5th larval instar, the relative abundance of fibroin RNA in the total RNA was fairly constant during the 4th and 5th instars. The increased content of fibroin RNA in 5th instar was the consequence of an overall increase in transcription accompanying the development progress, rather than specific increase only in fibroin transcription. The contents of fibroin protein in the 4th and 5th instars of development have also been quantitated making use of a sensitive radioimmune assay with a purified, antifibroin antibody. There were substantial differences between 4th and 5th instars in the absolute fibroin contents as well as the relative proportion of fibroin in the total proteins. These results implied that although the fibroin gene was transcribed at the same efficiency during the 4th and 5th instars, the translational efficiency was much lower during the 4th instar. The extent of polyadenylation of fibroin RNA was similar in both instars. However, there was a two-fold increase in the polysome association of fibroin RNA in the 5th instar. Over and above this, there was substantial increase during the 5th instar in the contents of those tRNAs. (e.g. Gly, Ala and Ser) which are abundantly represented in fibroin and therefore directly related to the expression of fibroin. The increased polysome association of fibroin mRNA and the adequate supply of cognate tRNAs in the 5th instar, together contributes to the translational regulation of fibroin in a developmental stage-specific manner. Based on these observations, we propose that translational regulation plays a major role in the development stage-specific synthesis of fibroin in Bombyx mori.

Animals↗
PubMed1984

Reduced level of secretion and absence of subunit combination for the fibroin synthesized by a mutant silkworm, Nd(2).

Fibroin is normally composed of one H chain (350 kd) and one L chain (25 kd) which are connected by disulfide bond(s). However, the small amount of fibroin secreted into the lumen of the posterior silk gland of the Nd(2) (naked pupa) mutant does not contain L chain, although L chain mRNA is present and L chain is synthesized in the posterior silk gland cells of the mutant. In a hybrid silkworm, Nd(2)/Tamanashikasuri, where Tamanashikasuri is a normal producer of fibroin, L chain from the two alleles are distinguishable electrophoretically. It is demonstrated using this system that the L chain from the Nd(2) allele can combine normally with the H chain from Tamanashikasuri and the H-L complex is secreted normally. In another hybrid system, Nd(2)/J-131, where J-131 is a normal producer of fibroin, fibroin derived from the two alleles are distinguishable due to the different electrophoretic mobility of H chain. The fibroin derived from the J-131 allele is composed of H chain and L chain, while the fibroin derived from the Nd(2) allele is devoid of L chain, and its secretion is greatly reduced. We present evidence suggesting that the H chain derived from the Nd(2) allele is structurally abnormal and discuss how the H-L subunit structure is advantageous in the secretion of fibroin.

Alleles↗
PubMed1975

Translation of silk fibroin messenger RNA in an Ehrlich ascites cell-free extract.

RNA identified by its base composition and T1 RNase oligonucleotide pattern as the message for silk fibroin was purified from mature posterior silk glands of Bombyx mori larvae and used to direct polypeptide synthesis in an Ehrlich ascites cell-free extract. Fibroin mRNA stimulated [3-H]alanine incorporation about 3- to 4-fold in the presence of 80 mM K+ and 4 mM Mg-2+. The stimulation was reduced in the presence of 5 times 10-minus 6 to 10-minus 4 M aurintricarboxylic acid, an inhibitor of the initiation of protein synthesis. The cell-free products were heterogeneous in size, including peptides as large as 100,000 daltons. They co-precipitated with carrier fibroin sequences after digestion with trypsin. A large fraction of the polypeptides synthesized in response to fibroin mRNA was precipitated by antiserum directed against amino acid sequences in noncrystalline region polypeptides of fibroin. Furthermore, after digestion with chymotrypsin, a major fraction of the cell-free products specifically co-precipitated with crystalline region sequences of native fibroin. The size and amino acid composition of the fibroin crystalline region polypeptides isolated from the cell-free products were similar to those from native fibroin.

Amino Acids↗