Ding J L, Navas M A, Ho B
Marine Biotechnology Laboratory, National University of Singapore, Kent Ridge.
Mol Mar Biol Biotechnol. 1995 Mar;4(1):90-103.
Two forms of Factor C cDNAs: CrFC21 (3448 bp) and CrFC26 (4182 bp) have been cloned into lambda gt22. CrFC26 includes 568 nucleotides of 5' untranslated region (5' UTR) containing seven ATGs before the real initiation site, an open reading frame (ORF) of 3249 nucleotides, a stop codon, and 365 nucleotides of 3' untranslated sequence. There are four polyadenylation signals and six potential glycosylation sites. The ORF codes for a signal peptide of 24 amino acids and a Factor C zymogen of 1059 residues. The CrFC21 lacks most of the 5' UTR, and has some base changes in its ORF. The predicted secondary mRNA structures of the 5' end of CrFC26 showed numerous stem-and-loop structures, thus obscuring its real start codon. In contrast, CrFC21 has a well-exposed AUG start site, and expresses Factor C in transcription-translation reactions in vitro. There is a typical serine protease catalytic triad of Asp-His-Ser, which is structurally like prothrombin, but catalytically more similar to trypsin. Although an overall homology of 97.7% was observed in comparison with the Tachypleus tridentatus Factor C (TtFC) cDNA, there were notable differences in the restriction sites and subtle base substitutions in the CrFC cDNA. The high degree of homology between Factor C from T. tridentatus and C. rotundicauda substantiates, at the molecular level, the proximity of these two species in the course of evolution. This finding contravenes the apparent disparities with respect to their morphology, ecological habitat, and taxonomical classification.
两种形式的C因子cDNA：CrFC21（3448 bp）和CrFC26（4182 bp）已被克隆到λgt22中。CrFC26包含568个核苷酸的5'非翻译区（5'UTR），在真正的起始位点之前有七个ATG，一个3249个核苷酸的开放阅读框（ORF），一个终止密码子，以及365个核苷酸的3'非翻译序列。有四个聚腺苷酸化信号和六个潜在的糖基化位点。该ORF编码一个24个氨基酸的信号肽和一个1059个残基的C因子酶原。CrFC21缺少大部分的5'UTR，并且其ORF中有一些碱基变化。CrFC26 5'端预测的二级mRNA结构显示出许多茎环结构，从而掩盖了其真正的起始密码子。相比之下，CrFC21有一个暴露良好的AUG起始位点，并在体外转录-翻译反应中表达C因子。有一个典型的天冬氨酸-组氨酸-丝氨酸丝氨酸蛋白酶催化三联体，其结构类似于凝血酶原，但催化作用更类似于胰蛋白酶。尽管与中国鲎C因子（TtFC）cDNA相比，总体同源性为97.7%，但CrFC cDNA的限制性位点和细微碱基取代存在显著差异。中国鲎和圆尾鲎的C因子之间的高度同源性在分子水平上证实了这两个物种在进化过程中的亲缘关系。这一发现与它们在形态、生态栖息地和分类学分类方面明显的差异相矛盾。
