F. Fase-Fowler

The amplified H circle of methotrexate-resistant leishmania tarentolae contains a novel P-glycoprotein gene.

Acquired resistance to methotrexate in Leishmania species is often associated with the amplification of H circles, 68 kb duplex DNA circles containing a 30 kb inverted repeat. We report here that the H circle of Leishmania tarentolae contains an open reading frame, ltpgpA, that has the attributes of P‐glycoproteins (large plasma membrane proteins known to extrude lipophilic drugs from mammalian cells). Although amplification of H circles is associated with proportionally increased levels of a 5.5 kb transcript of the ltpgpA gene, such methotrexate resistant mutants are not cross‐resistant to any of the drugs extruded by mammalian multi‐drug resistant cells. In Leishmania, ltpgpA is part of a gene family containing at least two other members. Sequences homologous to one of the nucleotide binding sites of ltpgpA are conserved in other kinetoplastida.

Direct and inverted DNA repeats associated with P-glycoprotein gene amplification in drug resistant Leishmania.

The H circle of Leishmania species contains a 30 kb inverted duplication separated by two unique DNA segments, a and b. The corresponding H region of chromosomal DNA has only one copy of the duplicated DNA. We show here that the chromosomal segments a and b are flanked by inverted repeats (198 and 1241 bp) and we discuss how these repeats could lead to formation of H circles from chromosomal DNA. Selection of Leishmania tarentolae for methotrexate resistance indeed resulted in the de novo formation of circles with long inverted duplication, but two mutants selected for arsenite resistance contained new H region plasmids without such duplications. One of these plasmids appears due to a homologous recombination between two P‐glycoprotein genes with a high degree of sequence homology. Our results show how the same DNA region in Leishmania may be amplified to give plasmids with or without long inverted duplications and apparently by different mechanisms.

Kinetoplast DNA of Trypanosoma evansi

We show here that the kinetoplast DNA (kDNA) networks from six Trypanosoma evansi strains differ from those of T. brucei by their lack of maxi-circles and absence of mini-circle sequence heterogeneity. The lack of maxi-circles is sufficient to account for the inability of T. evansi to multiply in tsetse flies, since this requires functional mitochondria containing maxi-circle gene products. Judged by restriction enzyme analysis, five of the six T. evansi strains contain mini-circles that differ less than 4% in sequence. This type A mini-circle is found in strains from East Africa, West Africa and South America. Another strain from East Africa contains a very different mini-circle (type B), which shows about the same degree of hybridization to type A mini-circles as to a mini-circle from T. brucei. We propose that the pronounced sequence heterogeneity of the mini-circles of T. brucei has arisen by recombination of strains that had diverged for long periods of time in reproductive isolation. We further propose that the homogeneous mini-circles of T. evansi (and T. equiperdum) reflect the inability of species to mate. This proposal implies that mini-circle heterogeneity indicates (infrequent) genetic exchange and that all kinetoplastid flagellates with heterogeneous mini-circles exchange DNA.

Characterization of DNA from Trypanosoma brucei and related trypanosomes by restriction endonuclease digestion.

Abstract We have digested non-kinetoplast DNA from various Trypanosoma strains with restriction endonucleases and analysed the fragment distribution by one-dimensional agarose gel electrophoresis. Visual inspection of ethidium-stained gels shows differences in banding pattern between DNAs from Trypanosoma brucei, Trypanosoma evansi and Trypanosoma equiperdum and even between different T. brucei strains, but not between three different antigenic variants derived from the same T. brucei cell. By blotting the DNA on to nitrocellulose strips the restriction endonuclease recognition sites around genes available in cloned form can be analysed by molecular hybridization and we demonstrate this for the gene which codes for one of the variant surface glycoproteins of T. brucei. The use of this method for strain classification is discussed. Renaturation analysis of T. brucei non-kinetoplast DNA shows that about 68% of this is present as single-copy DNA with a complexity of 2.5 × 107 base pairs, whereas the remainder consists of intermediate repetitive and highly repetitive DNA. The latter fraction contains a DNA which is cut by AluI into fragments of 180 base pairs, bands in CsCl at 1.690 g/cm3 and contains duplex circles heterogeneous in size and circles with duplex tails.

Further analysis of intraspecific variation in Trypanosoma brucei using restriction site polymorphisms in the maxi-circle of kinetoplast DNA

We have compared the maxi-circle kinetoplast DNA of 21 Trypanosoma brucei sp. stocks by analysis of restriction sites for nine restriction endonucleases. The analysis shows most of these stocks to have a maxi-circle sequence similar to that of 11 previously analysed stocks, with a difference of less than 3% between any two stocks. However, seven stocks stand out from the rest with at least two sites lost or gained for six of the nine restriction enzymes used. These seven distinctive stocks fall into two groups with some shared and some unique polymorphisms. One group had already been designated the kiboko group on the basis of isoenzyme patterns, but the relationship between nuclear markers and maxi-circle type is less clear-cut for the other group, designated sindo. Both groups seem to be in a wild animal-tsetse fly transmission cycle, with occasional infections in domestic stock, and may be reproductively isolated from the main T. brucei sp. population. The existence of the kiboko and sindo sub-groups shows that the maxi-circle is not shielded from evolutionary change. The lack of difference observed between the maxi-circles of the majority of T. brucei sp. stocks, including the gambiense and rhodesiense variants, must therefore reflect their close homology. Two geographical trends occur in T. brucei as a whole: (a) a trend in maxi-circle size, with increasing length of the variable region from West to East Africa, and (b) a greater frequency of certain restriction enzyme polymorphisms in East African stocks as compared to West African stocks.

Variations in maxi-circle and mini-circle sequences in kinetoplast DNAs from different Trypanosoma brucei strains.

We have compared a total of 30 recognition sites for eight restriction endonucleases on the 20-kilobase-pair maxi-circle of kinetoplast DNAs from five different Trypanosoma brucei strains. In addition to three polymorphic sites were have found a 5 kilobase-pair region that is not cleaved by any of the eight enzymes and that varies in size over 1 kilobase pair in the strains analysed. Mini-circles from these five strains, digested with endonuclease TaqI or MboII, yield very complex fragment patterns, showing that extensive mini-circle sequence heterogeneity is a common characteristic of these T. brucei strains. The size distribution of mini-circle fragments in these digests was identical for different clones of the 427 strain, but very different for mini-circles from different strains. These results show that maxi-circle sequence is conserved, whereas mini-circle sequence is not. Restriction digests of maxi-circles could be useful in determining how closely two Trypanosoma strains are related, whereas mini-circle digests can serve as sensitive tags for individual strains.

DNA circles with cruciforms from Isospora (Toxoplasma) gondii

We have isolated a closed circular duplex DNA fraction from the unicellular parasite Isospora (Toxoplasma) gondii and examined the purified DNA by electron microscopy. A major part of this circular DNA consists of 12-micron circles containing a cruciform with 0.5-micron tails. We also found 23-micron circles with the properties expected of head-to-tail dimers of the 12-micron circles. Some of these dimers have two cruciforms with 0.4-micron tails, some have one cruciform with 0.8-micron tails. When ethidium bromide was diffused into the DNA solution, circles with tails were replaced by twisted circles without tails. Direct mixing of the DNA with high ethidium bromide concentrations (5 micrograms/ml) gave rise to highly twisted circles with tails. This proves that the tailed circles are covalently continuous and indicates that ethidium bromide blocks branch migration. The 0.5-micron tails are part of a 1.7-micron palindrome, which was visualized by spreading denatured DNA under snap-back conditions. We argue that the cruciform is not present in vivo and that the 12-micron circles may represent the mitochondrial DNA of Toxoplasma.

Kinetoplast DNA from Trypanosoma vivax and T. congolense

We have analysed kinetoplast DNA (kDNA) of the African trypanosomes Trypanosoma vivax and T. congolense. The maxi-circles from these organisms resemble those of T. brucei in size, but only to a limited extent in sequence as judged from restriction enzyme digests and DNA X DNA hybridization. The kDNA networks of T. vivax have three distinguishing features: they contain the highest maxi-circle concentration of any kDNA (at least twice that of T. brucei); they contain the smallest mini-circles (465 bp) yet found thus far and the width of the kDNA nucleoid in thin sections is correspondingly small (55 nm against 91 nm for T. brucei); they contain a substantial fraction of mini-circle dimers.

Maxi-circles in the kinetoplast DNA of Trypanosoma mega.

Abstract We have analysed the kinetoplast DNA (kDNA) of Trypanosoma mega for the presence of DNA circles other than mini-circles and mini-circle oligomers. Degradation of networks with restriction endonucleases EcoRI, HhaI or PstI or S 1 nuclease yields minor bands on gels, in addition to the mini-circle bands. The combined molecular weight of the minor bands is 15–17 × 10 6 . Electron micrographs of kinetoplast DNA spread in a protein monolayer contain occasional circles longer than 3 μm. The majority class of these circles has a contour length corresponding to a mass of 16.1 × 10 6 D. We conclude that these maxi-circles, catenated into mini-circle networks, are an essential component of intact kinetoplast DNA of Trypanosoma mega .