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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↗

PubMed1995

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.

Amino Acid Sequence↗

PubMed1981

Initiation of fibroin biosynthesis. I. Isolation of nascent fibroin peptides from the posterior silk gland of Bombyx mori.

1. Peptidyl-tRNA was prepared from the posterior silk gland ribosomes of Bombyx mori on the fourth to fifth days of the fifth instar to explore the initiation process in fibroin biosynthesis. 2. The peptidyl-tRNA was hydrolyzed at an alkaline pH and the resulting nascent peptides were fractionated on Sephadex G-75 and Sephadex G-200 columns into twelve fractions. Each fraction was analyzed for amino acid composition. 3. The nascent peptides of smaller molecular size were rather rich in glutamic and aspartic acids. However, the amino acid composition of the nascent peptides gradually approached that of fibroin as their molecular size increased. 4. A comparison between the nascent peptides of smaller molecular size and the small subunit of fibroin was made in respect to amino acid composition and tryptic peptide map. Considerable similarity between these two proteins was observed. The implications of these results in relation to the initiation process in fibroin biosynthesis are discussed.

Amino Acid Sequence↗

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↗

PubMed1972