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

1.丝素H链和L链基因的转录调控：比较了丝素H链基因在不同转录状态下的染色质结构。最显著的观察结果是，转录活跃状态（5龄后部丝腺）和暂时不活跃状态（4龄蜕皮期后部丝腺）的5'上游区DNase I超敏感位点的分布模式相似，但与转录不活跃状态（5龄中部丝腺）的分布模式显著不同。从后部丝腺cDNA表达文库中分离出一个cDNA克隆，该克隆产生的DNA结合蛋白显示出对L2序列的亲和力，L2序列是H链和L链基因5'上游区的共同序列之一。该克隆的核苷酸序列与延伸因子（EF）1 γ的核苷酸序列具有高度相似性。2.丝心蛋白H-和L-链之间的结合位点：使用抗肽抗体研究H-和L-链之间的二硫键的位置，所述抗肽抗体针对含有负责H-和L-链中的结合的推定半胱氨酸残基的合成肽产生。在从反相FPLC柱洗脱的级分中鉴定出与两种抗体反应的一种胰凝乳蛋白酶肽级分。3.家蚕丝素蛋白分泌突变体的分析：Nd-s和Nd突变体均不能分泌丝素蛋白。在Nd-s中，在L-链基因的第3内含子中发生了重组，并产生了嵌合L-链分子。在Nd中，缺少其C末端区域的H链的降解产物积累在细胞质中。4.丝心蛋白L链和P25之间的关系：这两种蛋白质在后部丝腺中产生，显示出免疫学上无关。

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.