Remillard S P, Lai E Y, Levy Y Y, Fulton C
Department of Biology, Brandeis University, Waltham, MA 02254.
Gene. 1995 Feb 27;154(1):39-45. doi: 10.1016/0378-1119(94)00860-u.
One of two similar genes in the unicellular eukaryote Naegleria gruberi is shown to encode calcineurin B (CnB), the regulatory subunit of calcium-calmodulin-regulated protein phosphatase 2B. Over a span of 156 amino acids, excluding divergent N-termini, the encoded sequence shows 62% identity with vertebrate CnB, and also shows sequence elements specific, among calcium-binding proteins, to CnB. In contrast, the sequence shows only 23% identity with N. gruberi flagellar calmodulin. CNB mRNA is readily detected in amoebae; its abundance increases fourfold during differentiation to flagellates, reaches a peak at 50-70 min, when flagella are forming, and then declines. A genomic clone matches an expressed cDNA, except that it is interrupted by two phase I introns. The position of one intron, which separates the divergent N-terminal domain from the four calcium-binding domains (EF hands), is shared with a yeast CNB gene; the other is located in the central helix between the two pairs of calcium-binding loops; features that support an ancient origin. These introns, the first found in protein-coding genes of Naegleria, are flanked by characteristic splice junction sequences. N. gruberi CnB also shares similarities with recoverins. The finding in a protist of a CNB gene that contains two introns separating functional domains, shares similarities to recoverins and shows increased expression during differentiation is provocative. If the phylogeny of major groups derived from ribosomal RNA is accepted, Naegleria is among the earliest branching eukaryotes known to contain canonical pre-mRNA introns.
单细胞真核生物格氏耐格里变形虫（Naegleria gruberi）中的两个相似基因之一被证明编码钙调神经磷酸酶B（CnB），它是钙调蛋白调节的蛋白磷酸酶2B的调节亚基。在156个氨基酸的跨度内，不包括不同的N端，编码序列与脊椎动物CnB的同一性为62%，并且在钙结合蛋白中还显示出CnB特有的序列元件。相比之下，该序列与格氏耐格里变形虫鞭毛钙调蛋白的同一性仅为23%。CNB mRNA在变形虫中很容易检测到；其丰度在分化为鞭毛虫的过程中增加四倍，在鞭毛形成时的50 - 70分钟达到峰值，然后下降。一个基因组克隆与一个表达的cDNA匹配，只是它被两个I类内含子打断。其中一个内含子的位置将不同的N端结构域与四个钙结合结构域（EF手）分开，这与酵母CNB基因相同；另一个位于两对钙结合环之间的中央螺旋中；这些特征支持其古老的起源。这些内含子是在格氏耐格里变形虫的蛋白质编码基因中首次发现的，两侧是特征性的剪接连接序列。格氏耐格里变形虫CnB也与恢复蛋白有相似之处。在一种原生生物中发现一个包含两个分隔功能结构域的内含子、与恢复蛋白有相似之处且在分化过程中表达增加的CNB基因，这很有启发性。如果接受基于核糖体RNA的主要类群的系统发育，那么耐格里变形虫是已知含有典型前体mRNA内含子的最早分支真核生物之一。
