Sideris D C, Rampias T N, Fragoulis E G
University of Athens, Department of Biochemistry-Molecular Biology, Greece.
Insect Biochem Mol Biol. 2000 Feb;30(2):153-61. doi: 10.1016/s0965-1748(99)00112-5.
Two overlapping clones encoding for a ribonuclease from six-day-old larvae of the insect Ceratitis capitata (Cc-RNase) have been isolated by immunoscreening a cDNA library and by 5' RACE. The sequence of the Cc-RNase cDNA contains an open reading frame of 414 nucleotides encoding for a precursor protein of 138 amino acids long with a putative signal peptide consisting of 19 amino acids. The calculated M(r) of the mature protein was found to be 13.7 kDa. Multiple alignments of the deduced amino acid Cc-RNase sequence with other ribonucleases revealed an approximate 25% average identity. Despite the low percentage of identity, histidine and lysine residues which are essential for its catalytic activity, were found to be completely conserved. Furthermore, expression of the clone in E. coli resulted in the production of a recombinant product that showed strong immunoreactivity with anti-RNase specific antibodies. These results support the hypothesis that the identified clone encodes for a protein which is a new member of the RNase superfamily.
通过免疫筛选cDNA文库和5' RACE技术，已分离出两个重叠克隆，它们编码来自地中海实蝇六日龄幼虫的一种核糖核酸酶（Cc-RNase）。Cc-RNase cDNA的序列包含一个414个核苷酸的开放阅读框，编码一个138个氨基酸长的前体蛋白，带有一个由19个氨基酸组成的假定信号肽。成熟蛋白的计算分子量为13.7 kDa。推导的氨基酸Cc-RNase序列与其他核糖核酸酶的多重比对显示平均一致性约为25%。尽管一致性百分比很低，但发现对其催化活性至关重要的组氨酸和赖氨酸残基是完全保守的。此外，该克隆在大肠杆菌中的表达导致产生了一种重组产物，该产物与抗核糖核酸酶特异性抗体表现出强烈的免疫反应性。这些结果支持了所鉴定的克隆编码一种蛋白质的假设，该蛋白质是核糖核酸酶超家族的一个新成员。
