Comparison of maxicircle DNAs of Leishmania tarentolae and Trypanosoma brucei.

Abstract

The conserved portions of the maxicircle DNAs of Leishmania tarentolae and Trypanosoma brucei are organized in a basically colinear manner over a 15-to 17-kilobase region that is interrupted by two small less-homologous sequences. The most highly conserved regions are those encoding the 9S and 12S genes. An approximately 12-kilobase region directly upstream of the 12S gene in the L. tarentolae maxicircle showed no sequence homology with the T. brucei maxicircle and also was not transcribed. An approximately 6-kilobase region in the T. brucei maxicircle in the same relative location also showed no sequence homology with the L. tarentolae maxicircle. We propose that evolution of maxicircle DNA occurs mainly within.this "divergent region."The mtDNA or kinetoplast DNA (kDNA) of the kinetoplastid protozoa (1, 2) consists of a catenated network of minicircles and maxicircles. The minicircles comprise approximately 95% of the mass of the network, are usually heterogeneous in sequence, and are probably not transcribed (3-5). The maxicircles are present at 20-50 copies per network, are homogeneous in sequence, and are extensively transcribed (6-9). Maxicircle DNA contains the genes for the putative mitochondrial miniribosomal RNAs and also contains sequences homologous to several structural genes from yeast (10) and Zea mays (11). In the African trypanosome Trypanosoma brucei, the maxicircle appears to be evolving in a complex fashion involving both base substitutions and insertions/deletions of up to 1.5 kilobases (kb) in a region of the molecule termed the "silent" or "variable" region (12).Interspecific size variation of maxicircle DNA is much more pronounced, ranging from 3 to 17 kb (2). Conservation of the 9S and 12S gene sequences has been shown for several species (13)(14)(15)(16), and conservation of other maxicircle sequences has also been reported for T. brucei and Trypanosoma cruzi (17) (19). The purified maxicircle DNA was digested with Msp I and the fragments were separated in agarose and isolated by electroelution into dialysis bags. T. brucei kDNA was digested with HindIII and the 9.3-kb maxicircle A fragment (see Fig. 2) was isolated by electroelution after separation in agarose. The 6.5-kb B and the 6.1-kb C maxicircle HindIII fragments were obtained from the pBR322 clones, pTbM-2 and pTbM-1, described previously (16). HindIII-excised inserts were recovered by electroelution following electrophoresis through agarose. The Hha I subfragments, a and b, of the L. tarentolae maxicircle Msp I fragment 3 were cloned into the EcoRI site of pBR322 after treatment with the Klenow fragment of Escherichia coli DNA polymerase I and EcoRI linker addition. Insert DNA was isolated from EcoRI-digested plasmid by agarose electrophoresis and electroelution. The 6.6-kb EcoRI/BamHII fragment (see Fig. 6) of the L. tarentolae maxicircle was obtained from the pBR322 clone, pLtl20 (6). Purified 120 insert DNA was digested with Sau3A and the fragments were isolated by electroelution after separation in agarose. Control ...

Conserved repeats in the kinetoplast maxicircle divergent region of Leishmania sp. and Leptomonas seymouri

“…It was also shown that transcription of the 12S rRNA gene starts within the DR in T. brucei (Michelotti et al 1992) and L. seymouri (Vasil'eva et al 2004). However, no DR transcripts were revealed by Northern hybridization in L. tarentolae (Simpson et al 1982;Muhich et al 1983). Undoubtedly, DR transcription requires thorough investigation using more powerful methods.…”

“…The divergent region (DR) was initially described as a variable and presumably non-coding segment of the kinetoplast maxicircle (Borst et al 1980(Borst et al , 1982Stuart and Gelvin 1982;Muhich et al 1983;Maslov et al 1984). It proved to be resistant to cloning (Stuart and Gelvin 1982;Simpson 1986) and sequencing.…”

Structural organization of the maxicircle variable region ofTrypanosoma brucei: identification of potential replication origins and topoisomerase II binding sites

“…2D). In The sequence immediately 3' to repeat [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] GYA is characterized by a more even base composition and absence of short tandem repeats. However, the 3' portion of this region does contain two tandem copies of a longer (545 -700 nt) sequence (Figs 1 and 2B).…”

“…The region flanked by the ND5 and 12S rRNA genes from the T. brucei maxicircle varies in size between different isolates (13)(14)(15)(16)(17) and diverges in sequence among trypanosomatid species (11,12 (42). It has been proposed that the VR of the trypanosomatid maxicircle has a similar function (13).…”

“…However, the major area of divergence between species, and essentially all the divergence within species, occurs in the region between the ND5 and 12S rRNA genes. This region, known as the variable (or divergent) region (VR) varies in length and sequence content between species (11,12). The length of this region also varies between isolates of T. brucei and contains rapidly evolving repetitive DNA sequences (13)(14)(15)(16)(17).…”