Analysis of gene function concerning tissue-specific production and secretion of fibroin

1.丝素H链和L链基因转录调控：比较了丝素H链基因在不同转录状态下的染色质结构。最值得注意的是，在转录活跃状态(5龄后丝腺)和暂时不活跃状态(4龄后丝腺)之间，DNA酶I超敏感部位在5‘上游区域的分布相似，但这些模式与转录不活跃状态(5龄中丝腺)的模式明显不同。从后部丝腺基因表达文库中克隆出一个与L2序列有亲和力的结合蛋白，该序列是H链和L链基因5‘上游的共同序列之一。该克隆的核苷酸序列与延伸因子(EF)1伽马的核苷酸序列高度相似。2.丝素蛋白H-链和L链的结合部位：用抗丝素蛋白H-链和L链结合的半胱氨酸残基的抗肽抗体研究了H-链和L链之间二硫键的位置。从反相FPLC柱洗脱的组份中鉴定出一个与两种抗体反应的凝乳酶多肽组份。3.家蚕丝素分泌突变体分析：ND-S和ND突变体均不能分泌丝素。在ND-S中，L链基因第3内含子发生了重组，产生了嵌合的L链分子。在ND中，缺少C端区的H链降解产物聚集在细胞质中。4.丝素蛋白L链与P25的关系：后丝腺产生的这两种蛋白在免疫上是无关的。

1. Transcriptional regulation of fibroin H- and L-chain genes: Chromatin structures of fibroin H-chain gene in different transcriptional states were compared. The most notable observation was that distributions of DNase I-hypersensitive sites in the 5' upstream region were similar between transcriptionally active (5th instar posterior silk gland) and temporarily inactive (4th molting posterior silk gland) states but those patterns were remarkably different from that of the transcriptionally inactive (5th instar middle silk gland) state. A cDNA clone was isolated from the posterior silk gland cDNA expression library, which produced a DNA-binding protein showing affinity to the L2 sequence, i.e. one of common sequences in the 5' upstream regions of H- and L-chain genes. The nucleotide sequence of this clone exhibited high-level similarity to that of the elongation factor(EF)1gamma. 2. Binding site between fibroin H- and L-chains: Position of the disulfide bond between H- and L-chains was investigated using anti-peptide antibodies raised against synthetic peptides containing putative cysteine residues responsible for the binding in both H- and L-chains. One chymotryptic peptide fraction reacting with both antibodies was identified among fractions eleuted from a reversed phase FPLC column. 3. Analysis of fibroin secretion mutants of the silkworm: Both Nd-s and Nd mutants are unable to secrete fibroin. In Nd-s, a recombination had taken place in the 3rd intron of the L-chain gene and produced a chimeric L-chain molecule. In Nd, degradation products of H-chain which lacked its C terminal region were accumulated in the cytoplasm. 4. Relation between fibroin L-chain and P25: These two proteins produced in the posterior silk gland were shown to be immunologically unrelated.