Hoffman R T, Schmidt E R, Case S T
Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi 39216-4505, USA.
J Biol Chem. 1996 Apr 19;271(16):9809-15. doi: 10.1074/jbc.271.16.9809.
Chironomid salivary glands contain 40 cells dedicated to the synthesis of a relatively small ensemble of silk proteins. Glands in some species contain a special lobe composed of 4 cells distinguishable from the others. We have cloned a special lobe-specific cDNA from Chironomus thummi salivary glands. Northern blots of salivary gland RNA demonstrated that the cDNA hybridizes to a 2.5-kilobase transcript present only in the special lobe. In situ hybridization mapped the gene encoding this cDNA to region A2b on polytene chromosome IV, the locus of the special lobe-specific Balbiani ring a. The deduced amino acid sequence encodes a protein with a calculated molecular mass of 77 kDa and numerous potential glycosylation sites; it appears unrelated to other known chironomid silk proteins. Polyclonal antibody, raised against a cDNA-encoded fusion protein, reacted exclusively with a special lobe-specific 160-kDa silk protein. Lectin binding studies indicate that the immunoreactive 160-kDa protein contains both N- and O-linked glycan moieties. We conclude that glycosylation most likely contributes to the difference between calculated and apparent molecular masses and that this cDNA encodes the special lobe-specific silk protein previously described as ssp160 (Kolesnikov, N. N., Karakin, E. I., Sebeleva, T. E., Meyer, L., and Serfling, E. (1981) Chromosoma 83, 661-677).
摇蚊唾液腺含有40个专门用于合成相对少量丝蛋白的细胞。某些物种的唾液腺中有一个由4个细胞组成的特殊叶，与其他细胞不同。我们从嗜尸摇蚊唾液腺中克隆了一个特殊的叶特异性cDNA。唾液腺RNA的Northern印迹显示，该cDNA与仅存在于特殊叶中的2.5千碱基转录本杂交。原位杂交将编码该cDNA的基因定位到多线染色体IV上的A2b区域，即特殊叶特异性巴尔比亚尼环a的位点。推导的氨基酸序列编码一种计算分子量为77 kDa且有许多潜在糖基化位点的蛋白质；它似乎与其他已知的摇蚊丝蛋白无关。针对cDNA编码的融合蛋白产生的多克隆抗体仅与一种特殊的叶特异性160 kDa丝蛋白发生反应。凝集素结合研究表明，具有免疫反应性的160 kDa蛋白同时含有N-连接和O-连接的聚糖部分。我们得出结论，糖基化很可能导致了计算分子量和表观分子量之间的差异，并且该cDNA编码了先前被描述为ssp160的特殊叶特异性丝蛋白（Kolesnikov, N. N., Karakin, E. I., Sebeleva, T. E., Meyer, L., and Serfling, E. (1981) Chromosoma 83, 661-677）。
