Genomic organization of Trypanosoma brucei kinetoplast DNA minicircles.

The sequences of seven new Trypanosoma brucei kinetoplast DNA minicircles were obtained. A detailed comparative analysis of these sequences and those of the 18 complete kDNA minicircle sequences from T. brucei available in the database was performed. These 25 different minicircles contain 86 putative gRNA genes. The number of gRNA genes per minicircle varies from 2 to 5. In most cases, the genes are located between short imperfect inverted repeats, but in several minicircles there are inverted repeat cassettes that did not contain identifiable gRNA genes. Five minicircles contain single gRNA genes not surrounded by identifiable repeats. Two pairs of closely related minicircles may have recently evolved from common ancestors: KTMH1 and KTMH3 contained the same gRNA genes in the same order, whereas KTCSGRA and KTCSGRB contained two gRNA genes in the same order and one gRNA gene specific to each. All minicircles could be classified into two classes on the basis of a short substitution within the highly conserved region, but the minicircles in these two classes did not appear to differ in terms of gRNA content or gene organization. A number of redundant gRNAs containing identical editing information but different sequences were present. The alignments of the predicted gRNAs with the edited mRNA sequences varied from a perfect alignment without gaps to alignments with multiple mismatches. Multiple gRNAs overlapped with upstream gRNAs, but in no case was a complete set of overlapping gRNAs covering an entire editing domain obtained. We estimate that a minimum set of approximately 65 additional gRNAs would be required for complete overlapping sets. This analysis should provide a basis for detailed studies of the evolution and role in RNA editing of kDNA minicircles in this species.