Mallinson D J, Livingstone J, Appleton K M, Lees S J, Coombs G H, North M J
Department of Biological and Molecular Sciences, University of Stirling, UK.
Microbiology (Reading). 1995 Dec;141 ( Pt 12):3077-85. doi: 10.1099/13500872-141-12-3077.
The cattle protozoan parasite Tritrichomonas foetus has multiple forms of cysteine proteinases. To investigate their diversity, PCR and reverse transcriptase PCR were used to isolate genomic DNA and cDNA fragments, respectively, encoding different cysteine proteinases. Seven genes have been identified, TFCP3-6 from amplification of genomic DNA and TFCP7-9 from amplification of cDNA. Comparison of the predicted amino acid sequences indicates that the T. foetus enzymes are cathepsin-L-like rather than cathepsin-B-like in structure. However, there is considerable diversity among the proteinases. TFCP7 and TFCP8 are most similar to one another (78% identity), while TFCP3 and TFCP9 are the least closely related (30% identity). All but one of the genes are single-copy, the exception being TFCP3, which was present in multiple copies in one of the three isolates examined. Single transcripts were detected for each of the seven genes. TFCP8 was expressed at the highest levels, while transcripts for TFCP4 were only just detectable. In T. foetus F2, the strain from which the genomic DNA and mRNA were isolated, transcripts of the five other genes were present at intermediate levels. When two other isolates were compared with F2, differences in the expression of individual genes were apparent, with either one or two of them not expressed. In spite of these differences the major cysteine proteinases detected in the three isolates using substrate-SDS-PAGE appeared identical. The data show that the multiplicity of cysteine proteinases in T. foetus is due, in part at least, to the presence of multiple genes and that some of the genes encode cysteine proteinases which are not among the high-activity enzymes detected previously.
牛源原生动物寄生虫胎儿三毛滴虫具有多种形式的半胱氨酸蛋白酶。为了研究它们的多样性，分别使用聚合酶链反应（PCR）和逆转录聚合酶链反应（RT-PCR）分离编码不同半胱氨酸蛋白酶的基因组DNA和cDNA片段。已鉴定出7个基因，通过基因组DNA扩增得到TFCP3 - 6，通过cDNA扩增得到TFCP7 - 9。预测氨基酸序列的比较表明，胎儿三毛滴虫的这些酶在结构上类似于组织蛋白酶L，而非组织蛋白酶B。然而，这些蛋白酶之间存在相当大的差异。TFCP7和TFCP8彼此最为相似（同一性为78%），而TFCP3和TFCP9的亲缘关系最不密切（同一性为30%）。除一个基因外，其他所有基因均为单拷贝，例外的是TFCP3，在所检测的三个分离株之一中它以多拷贝形式存在。检测到这7个基因各自都有单转录本。TFCP8的表达水平最高，而TFCP4的转录本仅勉强可检测到。在分离出基因组DNA和mRNA的胎儿三毛滴虫F2株中，其他5个基因的转录本水平处于中等。当将另外两个分离株与F2株进行比较时，各个基因表达的差异很明显，其中有一两个基因不表达。尽管存在这些差异，但使用底物 - SDS - 聚丙烯酰胺凝胶电泳在这三个分离株中检测到的主要半胱氨酸蛋白酶看起来是相同的。数据表明，胎儿三毛滴虫中半胱氨酸蛋白酶的多样性至少部分是由于多个基因的存在，并且一些基因编码的半胱氨酸蛋白酶并不在先前检测到的高活性酶之中。
