Siri A, Carnemolla B, Saginati M, Leprini A, Casari G, Baralle F, Zardi L
Laboratory of Cell Biology, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy.
Nucleic Acids Res. 1991 Feb 11;19(3):525-31. doi: 10.1093/nar/19.3.525.
By sequencing cDNA clones which cover the complete coding region of human tenascin (TN), we have established its primary structure. This confirms that, as in the case of chicken, TN is mainly made up of three groups of sequences with a high homology to Epidermal Growth Factor (EGF) fibronectin (FN) type III repeat and fibrinogen. Furthermore, we have determined the amino-terminal sequence of the mature peptide directly on purified TN. The main differences with respect to the chicken TN molecule are that in the human there are 14 and half EGF-like repeats compared to 13 and half in the chicken and that, as previously reported, there are 15 FN-like repeats compared to 11 in the chicken. By Polymerase Chain Reaction (PCR) amplification we have also studied the different splicing patterns of the TN pre-mRNA in cultured cells. The results show the presence of at least four different isoforms containing different numbers of FN-like type III repeats. Using purified human TN as immunogen, we have obtained numerous monoclonal antibodies (Mabs) to TN. By screening a human melanoma cDNA library in the expression vector lambda gt11 with these Mabs and subsequently sequencing the insert of the positive clones, we have been able to localize, within the TN molecule, the epitopes recognized by two of these Mabs: BC-4, which recognizes an epitope within the EGF-like sequence and BC-2 which recognizes an epitope within the FN like type III repeats whose expression is regulated by alternative splicing of the TN pre-mRNA. Thus, while the Mab BC-4 may be useful in studies on TN distribution (since it recognizes all different TN isoforms) BC-2 may be useful in the study of the expression of particular TN isoforms generated by the alternative splicing of the TN primary transcript.
通过对覆盖人腱生蛋白（TN）完整编码区的cDNA克隆进行测序，我们确定了其一级结构。这证实，与鸡的情况一样，TN主要由三组与表皮生长因子（EGF）、纤连蛋白（FN）III型重复序列和纤维蛋白原有高度同源性的序列组成。此外，我们直接在纯化的TN上确定了成熟肽的氨基末端序列。与鸡TN分子的主要差异在于，人TN有14.5个EGF样重复序列，而鸡有13.5个；并且，如先前报道，人TN有15个FN样重复序列，而鸡有11个。通过聚合酶链反应（PCR）扩增，我们还研究了培养细胞中TN前体mRNA的不同剪接模式。结果显示存在至少四种不同的同工型，它们含有不同数量的FN III型重复序列。使用纯化的人TN作为免疫原，我们获得了许多针对TN的单克隆抗体（Mab）。通过用这些Mab筛选表达载体λgt11中的人黑色素瘤cDNA文库，并随后对阳性克隆的插入片段进行测序，我们得以在TN分子内定位其中两种Mab所识别的表位：BC-4识别EGF样序列内的一个表位，BC-2识别FN III型重复序列内的一个表位，其表达受TN前体mRNA可变剪接的调控。因此，虽然Mab BC-4可能在TN分布研究中有用（因为它识别所有不同的TN同工型），但BC-2可能在研究由TN初级转录本可变剪接产生的特定TN同工型的表达方面有用。
