Molecular cloning, regulation, and complete sequence of a hemocyanin-related, juvenile hormone-suppressible protein from insect hemolymph.
Molecular cloning, regulation, and complete sequence of a hemocyanin-related, juvenile hormone-suppressible protein from insect hemolymph.
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DOI：
10.1016/s0021-9258(19)38929-x
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发表时间：
1990-05
期刊：
影响因子：
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通讯作者：
G. Jones;N. Brown;M. Mańczak;S. Hiremath;F. Kafatos
中科院分区：
文献类型：
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作者：
G. Jones;N. Brown;M. Mańczak;S. Hiremath;F. Kafatos
从鳞翅目粉纹夜蛾幼虫-蛹变态期的mRNA中制备cDNA文库。差异探测用于鉴定可被外源保幼激素处理抑制的mRNA的克隆。在体外转录的cRNA从这些克隆在体外翻译和挑战与已知的酸性，青少年hemolymph蛋白（AJSP-1），这是与幼虫变态特异性的抗血清。发现三个克隆编码免疫反应性翻译产物，其身份通过比较成熟AJSP-1蛋白的N-末端序列与cDNA序列得到确认。从cDNA序列推断，该蛋白质包含704个氨基酸残基，包括N-末端信号肽;广泛分布以及更局部化的更强的序列相似性表明该蛋白质与血蓝蛋白和血蓝蛋白样昆虫蛋白质有较远的关系。然而，基于氨基酸序列和组成、免疫反应性和激素敏感性，该蛋白质不同于先前描述的昆虫蛋白质。其保幼激素抑制性可归因于mRNA的抑制。使用克隆的cDNA作为探针的RNA印迹分析表明，转录本（约2.8个酶）是非常低的丰度在倒数第二个体育场，但在最后的幼虫体育场，当保幼激素迅速下降变得非常丰富。此外，用保幼激素类似物处理幼虫强烈抑制了信息的丰度。
A cDNA library was prepared from mRNA isolated from the lepidopteran Trichoplusia ni during larval-pupal metamorphosis. Differential probing was used to identify clones for mRNAs which are suppressible by exogenous juvenile hormone treatment. In vitro transcribed cRNAs from these clones were translated in vitro and challenged with antiserum specific for a known acidic, juvenile hormone-suppressible hemolymph protein (AJSP-1) that is associated with larval metamorphosis. Three clones were found which encoded immunoreactive translation products; their identity was confirmed by comparison of the N-terminal sequence of the mature AJSP-1 protein with the cDNA sequence. As inferred from the cDNA sequence, the protein encompasses 704 amino acid residues, including a N-terminal signal peptide; widely distributed as well as more localized stronger sequence similarities indicate that the protein is distantly related to hemocyanins and hemocyanin-like insect proteins. However, on the basis of amino acid sequence and composition, immunological reactivity, and hormonal sensitivity, the protein is distinct from previously described insect proteins. Its juvenile hormone suppressibility can be ascribed to suppression of the mRNA. RNA blot analysis using the cloned cDNA as a probe demonstrated that the transcript (approximately 2.8 kilobases) is of very low abundance during the penultimate stadium but becomes very abundant during the last larval stadium, when juvenile hormone rapidly declines. Furthermore, treatment of larvae with a juvenile hormone analog strongly suppresses the abundance of the message.