Messina A, Neri M, Perosa F, Caggese C, Marino M, Caizzi R, De Pinto V
Istituto di Scienze Biochimiche e Farmacologiche, Facoltà di Scienze M.F.N., Università di Catania, Italy.
FEBS Lett. 1996 Apr 8;384(1):9-13. doi: 10.1016/0014-5793(96)00268-2.
We have raised polyclonal antibodies against purified the Drosphila melanogaster mitochondrial porin. They showed high titre and specificity and were thus used as a tool for screening an expression library. The isolated clone 1T1 showed 74% sequence identity in the last 19 residues at the C-terminus of human porin. A subclone of 1T1, containing the porin-like sequence, was thus used as a probe for re-screening a cDNA library and several positive clones were plaque-purified. We present here the sequence of a 1363 bp cDNA encoding a protein of 279 amino acids. Its identity with porin was also confirmed by N-terminal Edman degradation of the purified protein. The D. melanogaster porin shows an overall 51.8% identity with human porin isoform 1 (porin 31HL or HVDAC1) and an overall 55.7% identity with human porin isoform 2 (HVDAC2). Hydrophobicity plots and secondary structure predictions showed a very high similarity with data obtained from known porin sequences. The D. melanogaster porin cDNA was used as a probe for in situ hybridization to polytenic salivar gland chromosomes. It hybridizes with different intensities in two sites, in chromosome 2L, at region 31E and in chromosome 3L at region 79D. Thus, also in Drosophila melanogaster porin polypeptide(s) belong(s) to a multigene family.
我们制备了针对纯化的黑腹果蝇线粒体孔蛋白的多克隆抗体。这些抗体显示出高滴度和特异性，因此被用作筛选表达文库的工具。分离得到的克隆1T1在人孔蛋白C末端的最后19个残基中显示出74%的序列同一性。因此，1T1的一个包含孔蛋白样序列的亚克隆被用作探针重新筛选cDNA文库，几个阳性克隆被菌斑纯化。我们在此展示了一个1363 bp cDNA的序列，该cDNA编码一个279个氨基酸的蛋白质。通过对纯化蛋白进行N端埃德曼降解也证实了它与孔蛋白的同一性。黑腹果蝇孔蛋白与人类孔蛋白异构体1（孔蛋白31HL或HVDAC1）的总体同一性为51.8%，与人类孔蛋白异构体2（HVDAC2）的总体同一性为55.7%。疏水性图谱和二级结构预测显示与从已知孔蛋白序列获得的数据非常相似。黑腹果蝇孔蛋白cDNA被用作探针与多线唾液腺染色体进行原位杂交。它在两个位点以不同强度杂交，一个在2L染色体的31E区域，另一个在3L染色体的79D区域。因此，在黑腹果蝇中，孔蛋白多肽也属于一个多基因家族。
