Maxicircle (mitochondrial) genome sequence (partial) of Leishmania major: Gene content, arrangement and composition compared with Leishmania tarentolae

Abstract

We report 8420 bp of DNA sequence data from the maxicircle (mitochondrial) genome of Leishmania major (MHOM/SU/73/5ASKH), a much larger portion of this genome than has been reported previously from any Leishmania species infecting humans. This region contains 10 partial and complete genes: 5 protein-encoding genes (COII, COIII, ND1, ND7 and Cyt b); two ribosomal RNA subunits (12S and 9S) and three unidentified open reading frames (MURF1, MURF4 (ATPase6) and MURF5), as in the lizard-infecting species L. tarentolae. The genes from L. major exhibit 85–87% identity with those of L. tarentolae at the nucleotide level and 71–94% identity at the amino acid level. Most differences between sequences from the two species are transversions. The gene order and arrangement within the maxicircle of L. major are similar to those in L. tarentolae, but base composition and codon usage differ between the species. Codons assigned for initiation for protein-coding genes available for comparison are similar in five genes in the two species. Pre-editing was identified in some of the protein-coding genes. Short intergenic non-coding regions are also present in L. major as they are in L. tarentolae. Intergenic regions between 9S rRNA and MURF5, MURF1 and ND1 genes are G+C rich and considered to be extensive RNA editing regions. The RNA editing process is likely to be conserved in similar pattern in L. major as in L. tarentolae.

Introduction

Leishmania major belongs to the order Kinetoplastida, family Trypanosomatidae, members of which all contain a kinetoplast situated at the base of the flagellum.The kinetoplast contains a concatenated network of circular DNA molecules, the kinetoplast DNA (kDNA) (Shlomai, 2004), comprised of 20–50 maxicircles and about 10,000 minicircles (Liu et al., 2005).

Maxicircles are circular DNA molecules, 20–35 kb in size, and have many of the characteristics of conventional mitochondrial DNA. All copies are similar in composition, encoding proteins involved in energy production and ribosomal RNAs (Maslov et al., 1984). The DNA sequence of the mitochondrial maxicircle of Leishmania tarentolae (infecting lizards)(de la Cruz et al., 1984, de la Cruz et al., 1985, Simpson et al., 1987) has been reported and six structural genes identified (the cytochrome oxidase subunits COI, COII, and COIII, cytochrome b (Cyt b) and NADH dehydrogenase subunits 4 (ND4) and 5 (ND5)). Three other open reading frames, referred to as MURF1, MURF2, and MURF4 (now known to be ATPase6 (Bhat et al., 1990)) as well as RSP12, 12S and 9S rRNA genes have also been identified in L. tarentolae. Complete maxicircle sequences of Trypanosoma brucei (Benne et al., 1983, Eperon et al., 1983, Payne et al., 1985, Hensgens et al., 1984, Johnson et al., 1984, Feagin et al., 1985, Hong and Simpson, 2003) and Trypanosoma cruzi (Ochs et al., 1996, Westenberger et al., 2006) are also available. Partial maxicircle sequences are available for a number of other kinetoplastids: Trypanosoma congolense (L16531), Trypanosoma equiperdum (U03741), Trypanoplasma borreli (U11682), Bodo saltans, Crithidia fasciculata (X15081) and Phytomonas serpens (AF079967) (e.g. Maslov et al., 1984, Feagin, 2000). In the maxicircle of trypanosomes, the genes are tightly clustered, and all (12S rRNA, 9S rRNA, ND8, ND7, COIII, Cyt b, MURF4 (ATPase 6), COII, MURF2, ND4, RSP12, ND5) are transcribed from the same DNA strand as the ribosomal genes, except for ND3, COI, ND1, MURF1 and MURF5 (de la Cruz et al., 1984, Ochs et al., 1996). The six identified structural genes of L. tarentolae showed various degrees of divergence from the homologous genes in other kinetoplastids, with COI the most conserved and COIII the least conserved (de la Cruz et al., 1984). Maxicircle genes are often incomplete and may lack a start codon (ATG) (Borst et al., 1980). Post–transcriptional uridine insertion/deletion RNA editing resolves most of these problems, by creating start codons (Feagin et al., 1988a, Shaw et al., 1988, Myler et al., 1993), correcting internal frame shifts (e.g., four uridines are inserted in COII) (Payne et al., 1985, Benne et al., 1986, Shaw et al., 1989) and extensively modifying unrecognizable mRNA transcripts to create entire ORFs (van der Spek et al., 1988, Shaw et al., 1989) (e.g.547 uridines are inserted and 41 deleted in COIII of Trypanosoma brucei) (Feagin et al., 1988b). The process of RNA editing has been intensively studied in Trypanosoma brucei, T. cruzi, L. tarentolae and Crithidia fasciculata (Simpson et al., 2003, Stuart and Paniigrahi, 2002). The maxicircle genome of all trypanosomatids contain an unusually long non-coding region (from 5–20 kb), also termed the divergent region (DR). Some research groups have found promoters for the 12S rRNA gene and for minicircle replication (CSB-I, II, III) (Horvarth et al., 1990, Vasil’eva et al., 2004) within this region.

Minicircles, on the other hand are heterogeneous in sequence and make up the bulk of the kDNA mass with tens of thousands of copies per network. They carry the specific information for RNA editing in the form of guide RNA (gRNA) molecules that function in editing of maxicircle mRNA transcripts (Borst et al., 1980, Maslov et al., 1994, Simpson et al., 2003). The gRNAs interact with the mRNA templates through hybridization to dictate the precise location and number of uridine insertions or deletions to be made during RNA editing (Simpson et al., 2003).

Leishmania major, an important pathogen which causes cutaneous (localized and diffuse) lesions is endemic to North Africa, Middle East and Western India (Desjeux, 2004). In most cases, multiple lesions develop on exposed parts of the body, often on the face, that usually heal in a few months but occasionally last for many years, causing considerable morbidity and large scars (Desjeux, 2004). The nuclear genome of L. major (Friedlin strain) has been sequenced and found to contain 911 RNA genes, 39 pseudogenes and 8272 protein-coding genes (Ivens et al., 2005). However, to date, only a few maxicircle genes of this species have been sequenced and identified. These are the cytochrome b gene (AB095970, AB095961), cytochrome c oxidase II (EF633106), a sequence similar to NADH dehydrogenase subunit 1 (AF395133) and partial maxicircle control region sequence extending to the 12S ribosomal RNA gene (DQ107358). Partial but extensive DR sequence of L. major has also been obtained by Flegontov et al. (2006). In this study, we report the sequence of about 8.4 kb from the coding region of the L. major maxicircle genome and present a comparative analysis of structure, gene content and genetic composition between the two Leishmania species, L. major and L. tarentolae. Our data constitute a much larger portion of the maxicircle genome than has been reported previously from L. major or any other Leishmania species infecting humans. The main objective of this study was to provide a rich source of information for genotyping and diagnosis, phylogenetic, evolutionary and epidemiological studies of the genus Leishmania (e.g. Fernandes et al., 1999, Brewster and Barker, 1999, Luyo-Acero et al., 2004). Mitochondrial genes also present possible drug targets for disease intervention: atovaquone, a drug used against toxoplasmosis and malaria is a good example (Feagin, 2000). This data will also provide the factors that are unique to the Leishmania genus that can be used as potential drug targets or vaccine candidates.