Guide RNAs of the recently isolated LEM125 strain of Leishmania tarentolae: An unexpected complexity

Guide RNAs (gRNAs) are encoded both in the maxicircle and minicircle compon ents of the mitochondrial DNA of trypanosomatid protozoa. These RNAs mediat e the precise insertion and deletion of U residues in transcripts of the ma xicircle DNA. We showed previously that the old UC laboratory strain of Lei shmania tarentolae apparently lost more than 40 minicircle-encoded gRNAs th at are present in the recently isolated LEM125 strain [Thiemann et al., EMB O J, 1994, 13:5689-5700]. We have further analyzed the population of minici rcle-encoded gRNAs in the LEM125 strain. Sau3AI and MspI minicircle librari es were constructed and screened for novel gRNAs by negative colony hybridi zation. This search yielded 20 minicircles encoding new gRNAs that covered most of the remaining gaps in the editing cascades of the ND8, ND9, G4, and G5 genes, and in addition, more than 30 minicircles containing either unas signed or undetectable gRNA genes. We also completely sequenced 34 of the 4 5 minicircle sequence classes encoding previously identified gRNAs. A total of 19 pairs of redundant gRNAs, which are gRNAs of different sequences cov ering the same editing blocks, were identified. The gRNAs in each redundant pair generally had different relative abundances and different extents of mismatches with edited sequences. Alignments of the minicircles encoding re dundant gRNAs yielded 59 to 93% matching nucleotides, suggesting an origin from duplication of ancestral minicircles and subsequent genetic drift. We propose a functional explanation for the existence of redundant gRNAs in th is strain.