Redpath M B, Carnall N, Webb H, Courel M, Amorim A, Güther M L, Cardoso de Almeida M L, Carrington M
Department of Biochemistry, University of Cambridge, UK.
Mol Biochem Parasitol. 1998 Jul 1;94(1):113-21. doi: 10.1016/s0166-6851(98)00056-5.
The experiments described in this paper were designed to try and isolate a recombinant DNA clone encoding a Trypanosoma cruzi homologue of the Trypanosoma brucei glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) gene. Despite the ready biochemical detection of phospholipase C activities that hydrolyse GPI-anchors of cell surface proteins in T. cruzi, it did not prove possible to isolate any recombinant DNA clones using the T. brucei gpi-plc gene as a probe. On determining the DNA sequence to the 5' side of the gpi-plc gene it was found to be adjacent to a gene that encodes a 100 kDa heat shock protein (HSP100). To investigate whether this linkage between the hspl00 and gpi-plc genes was conserved in T. cruzi, a probe derived from the T. brucei hsp100 gene was used to isolate T. cruzi genomic clones. These were partially sequenced and shown to contain an hsp100 gene. Restriction enzyme fragments located to the 3' side of the T. cruzi hsp100 gene were then sequenced and found to contain a gene that encodes a polypeptide (TcPLC1) that has 46% amino acid sequence identity with the T. brucei GPI-PLC including most of the key residues involved in inositol binding and the catalytic histidine. A recombinant form of TcPLC1 was produced and shown to possess phospholipase C activity towards a GPI-substrate. Thus, the hsp100 and gpi-plc genes are adjacent in T. brucei and this linkage is conserved in T. cruzi. This observation has been used to facilitate the isolation of a clone encoding a T. cruzi phospholipase C gene.
本文所述实验旨在尝试分离出一个重组DNA克隆，该克隆编码与布氏锥虫糖基磷脂酰肌醇特异性磷脂酶C（GPI-PLC）基因同源的克氏锥虫基因。尽管在克氏锥虫中能够轻易通过生化方法检测到水解细胞表面蛋白GPI锚定物的磷脂酶C活性，但使用布氏锥虫gpi-plc基因作为探针却未能分离出任何重组DNA克隆。在确定gpi-plc基因5'端的DNA序列时，发现它与一个编码100 kDa热休克蛋白（HSP100）的基因相邻。为了研究hsp100和gpi-plc基因之间的这种连锁关系在克氏锥虫中是否保守，使用了源自布氏锥虫hsp100基因的探针来分离克氏锥虫基因组克隆。对这些克隆进行了部分测序，结果显示它们含有一个hsp100基因。然后对位于克氏锥虫hsp100基因3'端的限制性酶切片段进行测序，发现其中包含一个编码多肽（TcPLC1）的基因，该多肽与布氏锥虫GPI-PLC具有46%的氨基酸序列同一性，包括参与肌醇结合的大多数关键残基和催化组氨酸。制备了重组形式的TcPLC1，并证明它对GPI底物具有磷脂酶C活性。因此，hsp100和gpi-plc基因在布氏锥虫中相邻，并且这种连锁关系在克氏锥虫中保守。这一发现已被用于促进编码克氏锥虫磷脂酶C基因的克隆的分离。
