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.     

Expression of the hGM-CSF in the silk glands of germline of gene-targeted silkworm

To express human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene in the silk glands of transformation silkworm (Bombyx mori) based on gene-targeting, two fragments from fibroin heavy chain gene (fib-H) of silkworm were cloned and sequenced. One fragment contains the 1st exon and its downstream 1st intron’s partial sequence; and the other fragment contains the 1st intron’s partial sequence and the 2nd exon’s partial sequence. Then the two fragments, as homologous arm, were inserted into pSK to generate a gene-targeted vector, pSK-HL-A3GFP-FLP-GM-CSF-FLPA-HR in which a gfp gene driven by A3 promoter and an hGM-CSF gene under the control of fibroin light chain (fib-L) promoter were included. The vector was transferred into the silkworm eggs using sperm-mediated gene transfer. After being screened for green fluorescent, the transformation silkworm was obtained, whose genome was verified by PCR and dot hybridization to confirm whether the target genes had been integrated into the silkworm genome. Furthermore, in the posterior silk glands of the G4 generation transformation silkworms, a specific band with the molecular weight of 22 kDa could be detected by Western blotting with an antibody against hGM-CSF, and the expression level of the hGM-CSF estimated by ELISA was approximately 1.26 ng per gram fresh posterior silk gland.     

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.     

Carbonic anhydrase generates a pH gradient in Bombyx mori silk glands

Silk is a protein of interest to both biological and industrial sciences. The silkworm, Bombyx mori, forms this protein into strong threads starting from soluble silk proteins using a number of biochemical and physical cues to allow the transition from liquid to fibrous silk. A pH gradient has been measured along the gland, but the methodology employed was not able to precisely determine the pH at specific regions of interest in the silk gland. Furthermore, the physiological mechanisms responsible for the generation of this pH gradient are unknown.In this study, concentric ion selective microelectrodes were used to determine the luminal pH of B. mori silk glands. A gradient from pH 8.2 to 7.2 was measured in the posterior silk gland, with a pH 7 throughout the middle silk gland, and a gradient from pH 6.8 to 6.2 in the beginning of the anterior silk gland where silk processing into fibers occurs. The small diameter of the most anterior region of the anterior silk gland prevented microelectrode access in this region. Using a histochemical method, the presence of active carbonic anhydrase was identified in the funnel and anterior silk gland of fifth instar larvae. The observed pH gradient collapsed upon addition of the carbonic anhydrase inhibitor methazolamide, confirming an essential role for this enzyme in pH regulation in the B. mori silk gland. Plastic embedding of whole silk glands allowed clear visualization of the morphology, including the identification of four distinct epithelial cell types in the gland and allowed correlations between silk gland morphology and silk stages of assembly related to the pH gradient.B. mori silk glands have four different epithelial cell types, one of which produces carbonic anhydrase. Carbonic anhydrase is necessary for the mechanism that generates an intraluminal pH gradient, which likely regulates the assembly of silk proteins and then the formation of fibers from soluble silk proteins. These new insights into native silk formation may lead to a more efficient production of artificial or regenerated silkworm silk fibers.     

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.     

Novel enhancer and promoter elements indispensable for the tissue-specific expression of the sericin-1 gene of the silkworm Bombyx mori

Sericins are glue proteins produced specifically in the middle silk gland (MSG) of the silkworm Bombyx mori, while the silk fiber protein, fibroin, is produced in the posterior silk gland (PSG). These silk proteins are expected to be useful biomaterials in medical technology as well as biotechnology. In this study, we analyzed promoter elements of the sericin-1 gene (ser1) in vivo by introducing reporter constructs into silk glands via gene gun technology. The region from −1602 to +47 was sufficient to induce MSG-specific expression. The 5′ deletion mutants showed a three-step decrease in promoter activity with the key sequences located between −1362 and −1250, −201 and −116, and −115 and −37. We detected a tissue- and stage-specific factor complex (MSG-intermolt-specific complex: MIC) bound to the sequence elements around the −1350, −320, −180, and −70 regions. A mutation in the −70 region, which inhibits MIC-binding, diminished almost all promoter activity, while another mutation that did not inhibit MIC-binding showed no effect on promoter activity. The results suggest that the binding of MIC to the above elements is intrinsic for the spatiotemporal specificity of ser1 in vivo.