Eukaryotic protein-coding genes are generally transcribed by RNA polymerase II (Pol II), which has a lower transcription rate than that of Pol I. We report here the duplication of two LD1 genes into the rRNA locus and their resultant transcription by Pol I. The multigenic LD1 locus is present in a 2.2-Mb chromosome in all stocks of Leishmania spp. and is also present in multicopy 200-to 450-kb linear chromosomes or multicopy circular DNAs in over 15% of stocks examined. Genomic rearrangement in Leishmania donovani LSB-51.1 resulted in duplication of a 3.9-kb segment of LD1 containing two genes (orfF and orfG) and of a 1.3-kb segment from ϳ10 kb downstream into the rRNA gene repeat region of the 1.2-Mb chromosome. Short sequences (12 or 13 bp) common to the 2.2-Mb LD1 and 1.2-Mb rRNA loci suggest that this gene conversion occurred by homologous recombination. Transcription of the duplicated genes is ␣-amanitin resistant, indicating transcription by Pol I, in contrast to the ␣-amanitin-sensitive (Pol II) transcription of the genes in the 2.2-Mb LD1 locus. This results in higher transcript abundance than expected from the gene copy number in LSB-51.1 and in elevated expression of at least the orfF gene product.Several multigene loci undergo amplification in Leishmania spp. (for a review, see reference 3). These gene amplifications have often been the result of selection for drug resistance in vitro (4,5,21,29,37). However, unselected strains of Leishmania spp. also have amplified DNAs, including LD1 (27, 52), but their significance is unknown. The amplicons are circular or linear molecules of various sizes and copy numbers, and the mechanism of their amplification appears to involve homologous recombination based on the presence of direct or inverted repeats at the termini of the amplified sequence. For example, rearrangement of the H region of Leishmania tropica has been shown to involve either direct or inverted DNA repeats of approximately 200 bp or greater (38, 55), while the circular LD1 (CD1) of Leishmania mexicana M379 is thought to be generated by recombination involving short (13-bp) direct repeats (28).We have been investigating the multigene LD1 locus that is present within a 2.2-Mb chromosome in Leishmania donovani and a similar-sized chromosome in all Leishmania strains. LD1 locus sequences are amplified as 20-to 60-copy, 200-to 450-kb chromosomes in some strains or 100-to 200-copy circular DNAs in others (52, 53). The LD1 locus contains nine genes, including one (orfB) which encodes ribosomal protein L37 (35), one (orfC) with homology to genes which appear to have a role in regulation of cell growth (36), and another (orfG) with homology to Trypanosoma brucei ESAG10 (18, 34). ESAG10 and orfG predict proteins with 10 to 12 potential membranespanning domains, indicating possible transporter function (34).In the Kinetoplastida, most protein-coding genes are transcribed as polycistronic units by RNA polymerase II (Pol II), similar to other eukaryotic systems. However, in T. brucei, the variant surface gly...