A CALCINEURIN-B-ENCODING GENE EXPRESSED DURING DIFFERENTIATION OF THEAMEBOFLAGELLATE NAEGLERIA-GRUBERI CONTAINS 2 INTRONS

One of two similar genes in the unicellular eukaryote Naegleria gruber i is shown to encode calcineurin B (CnB), the regulatory subunit of ca lcium-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 sp ecific, among calcium-binding proteins, to CnB. In contrast, the seque nce 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, wh en 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 wit h a yeast CNB gene; the other is located in the central helix between the two pairs of calcium-binding loops; features that support an ancie nt origin. These introns, the first found in protein-coding genes of N aegleria ia, are flanked by characteristic splice junction sequences. N. gruberi CnB also shares similarities with recoverins. The finding i n a protist of a CNB gene that contains two introns separating functio nal domains, shares similarities to recoverins and shows increased exp ression during differentiation is provocative. If the phylogeny of maj or groups derived from ribosomal RNA is accepted, Naegleria is among t he earliest branching eukaryotes known to contain canonical pre-mRNA i ntrons.