Isolation and characterization of kinetoplast DNA from Leishmania tarentolae

Simpson, Larry; Silva, Agda da

doi:10.1016/0022-2836(71)90394-9

Cited by 100 publications

(26 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Isolation of DNA from Leishmania promastigotes was performed as described (11); nuclear DNA was separated from kinetoplast DNA by centrifugation in CsCl/ethidium bromide density gradients (12). Restriction endonucleases were obtained from commercial sources and used as described (13).…”

Section: Methodsmentioning

confidence: 99%

Overproduction of a bifunctional thymidylate synthetase-dihydrofolate reductase and DNA amplification in methotrexate-resistant Leishmania tropica.

Coderre¹,

Beverley²,

Schimke³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

192

View full text Add to dashboard Cite

Leishmania tropica promastigotes that are highly resistant to methotrexate, a dihydrofolate reductase inhibitor, have been developed. Organisms resistant to 1 mM methotrexate have a 40-fold increase in dihydrofolate reductase which is associated with thymidylate synthetase, and they contain amplified regions of DNA that may be directly visualized on stained gels of restriction digests. The amplified DNA in these organisms is about 56 Idlobases in length, has a copy number about 80-fold higher than that of wild-type organisms, and constitutes about 10% of the nuclear DNA. When the methotrexate-resistant L. tropica are propagated in drug-free medium, the dihydrofolate reductase-thymidylate synthetase protein and the amplified DNA decrease in a parallel fashion until they are indistinguishable from the levels in wild-type organisms. However, when these apparent revertants are again challenged with 1 mM methotrexate, enzyme overproduction and DNA amplification occur rapidly. As in mammalian cells, it appears that drug resistance in parasitic protozoa may be mediated by gene amplification.Drug resistance in protozoan parasites was first described early in this century by Paul Ehrlich in his studies on the chemotherapy of trypanosomiasis (1). Since then, numerous instances of drug-resistant Trypanosoma (2, 3), Leishmania (3), and Plasmodium (4) have been documented. There are relatively few drugs that are curative agents for the diseases caused by pathogenic protozoa, and the emergence of drug-resistant organisms is a particularly serious problem. Furthermore, because knowledge of the mechanisms of drug resistance in protozoan parasites is limited, no rational approaches exist for circumventing or avoiding this problem.We have embarked upon investigations to define the molecular bases of drug resistance in pathogenic protozoa. In this report, we describe the development and properties of methotrexate (MTX)-resistant strains of Leishmania tropica promastigotes. We show that resistant organisms overproduce the drug target-a bifunctional dihydrofolate reductase (DHFR)-thymidylate synthetase (TS) enzyme-and amplify certain regions of DNA. These properties are unstable when selective pressure of MTX is removed but rapidly reemerge when organisms are reexposed to normally lethal concentrations of the drug. Last, we show that only a 10-fold increase in gene copy number in these protozoa may be detected by direct visualization of ethidium bromide-stained gels of DNA restriction digests.MATERIALS EC50 values refer to the concentration of MTX that inhibited the growth rate by 50%; unless otherwise specified, one passage refers to five generations of growth, or a 32-fold expansion in cell number, followed by reseeding in fresh medium. Cells were counted by using a Coulter Counter ZBI.Selection of MTX-Resistant Cel Lines. MTX-resistant strains of L. tropica promastigotes were obtained by using a stepwise selection process. Cells were seeded at approximately 106/ml in medium containing the specified amount of MTX. When th...

show abstract

Section: Methodsmentioning

confidence: 99%

Overproduction of a bifunctional thymidylate synthetase-dihydrofolate reductase and DNA amplification in methotrexate-resistant Leishmania tropica.

Coderre¹,

Beverley²,

Schimke³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

192

View full text Add to dashboard Cite

show abstract

“…Free mini-circles are virtually absent in this fraction, in agreement with the electrophoretic analysis presented in Fig. 5 and 6. c) The highly characteristic rosette structures present in intact networks and in networks degraded by shear or non-specific deoxyribonucleases [2,12,19] are absent in kDNA digested with endonucleases HindII + 111 (see Fig. 9).…”

Section: Electron Microscopy Of the Fractions Of Kdna Sensitive And Rmentioning

confidence: 99%

“…Biol. (1973) 81, 419-4231. of mini-circles [7,; long loops extending from the edge of the network and up to 12 pm long [3,4,6, 141; long linear DNA [4,[6][7][8]12,. Whether all these components are intrinsic parts of the network rather than contaminants has not been rigorously established.…”

mentioning

confidence: 99%

The Structure of Kinetoplast DNA

Kleisen

Borst

Weijers

1976

European Journal of Biochemistry

View full text Add to dashboard Cite

We have analysed limit digests of mini-circles from kinetoplast DNA of Crithidia Zuciliae by gel electrophoresis. Endonucleases Hap11 and AluI cut the circles into at least 37 and 21 fragments, respectively, and leave no circles intact. In both cases the added molecular weights of the fragments, cstirnated from mobility in gels, exceeds 18 x lo6, i.e. more than 12 times the molecular weight of the mini-circle DNA. Endonucleases HindII + 111, EcoRI and HpaI cut only part of the circles.These results show that the mini-circles are heterogeneous in base sequence. Different sequence classes are present in different amounts.DNA-DNA renaturation analysis of mini-circle DNA yields a complexity of about 3 x lo6, i.e. twice the molecular weight of one mini-circle. The At,, of native and renatured duplexes is about 1 "C, showing that the sequence heterogeneity is a micro-heterogeneity.Electron microscopy, gel electrophoresis and sedimentation analysis show that the circles that are not cut by endonucleases HindII + 111 remain catenated in very large associations. These associations lack the 'rosette' structures and the long edge loops characteristic of intact kinetoplast DNA.This suggests that the mini-circle classes cut by endonucleases HindII + I11 are present throughout the network and that the maxi-circle component of the network (see accompanying paper) is not essential to hold the network together. agarose gels resolves the open mini-circles into three and linearized mini-circles into four bands, present in different amounts. We conclude that the minicircles are also heterogeneous in size, the difference in size between the two extreme size classes being 4'x of the contour length. Digestion with endonuclease HupII shows that at least three out of these four bands differ in sequence.Possible mechanisms that could account for the micro-heterogeneity in sequence of minicircles are discussed.Prolonged electrophoresis on 1.5 %, or 2The kinetoplast DNA (kDNA) of the hemoflagellates is a complicated network of catenated molecules, with an overall molecular weight of about lo1' and is located in the single mitochondrion of these organisms [l]. This network contains as a main component about 1 O4 mini-circles which are extensively catenated and vary in size from 0.3 pm in Leishmania to 0.8 pm in Crithidia species [l -91. In addition to mini-circles, four types of longer molecules have been observed in isolated kDNA : circular molecules interpreted as circular oligomers or fused dimers Enzymes. S, nuclease from Aspergillus oryzue (EC 3.1.4.21); restriction endonucleases (EC 3.1.4. -) are named according to the suggestions of Smith and Nathans [J. Mol. Biol. (1973) 81, 419-4231. of mini-circles [7,; long loops extending from the edge of the network and up to 12 pm long [3,4,6, 141; long linear DNA [4,[6][7][8]12,. Whether all these components are intrinsic parts of the network rather than contaminants has not been rigorously established.A second problem still unsettled is the exact genetic information that is encoded in k...

show abstract

“…Different forms of catenation of minicircles in the network have been suggested on the basis of electronmicroscopic studies (1,26). By using topoisomerase, the kDNA network can be unlinked into monomeric maxicircles and minicircles (4,16,21).…”

mentioning

confidence: 99%

Selection for arsenite resistance causes reversible changes in minicircle composition and kinetoplast organization in Leishmania mexicana.

et al. 1994

View full text Add to dashboard Cite

Certain minor minicircle sequence classes in the kinetoplast DNA (kDNA) networks of arsenite-or tunicamycin-resistant Leishmania mexicana amazonensis variants whose nuclear DNA is amplified appear to be preferentially selected to replicate (S. T. Lee, C. Tarn, and K. P. Chang, Mol. Biochem. Parasitol. 58:187-204, 1993). These sequences replace the predominant wild-type minicircle sequences to become dominant species in the kDNA network. The switch from wild-type-specific to variant-specific minicircles takes place rapidly within the same network, the period of minicircle dominance changes being defined as the transition period. To investigate the structural organization of the kDNA networks during this transition period, we analyzed kDNA from whole arsenite-resistant Leishmania parasites by dot hybridization with sequence-specific DNA probes and by electron-microscopic examination of isolated kDNA networks in vitro. Both analyses concluded that during the switch of dominance the predominant wild-ype minicircle class was rapidly lost and that selective replication of variant-specific minicircles subsequently filled the network step by step. There was a time during the transition when few wild-type-or variant-specific minicircles were present, leaving the network almost empty and exposing a species of thick, long, fibrous DNA which seemed to form a skeleton for the network. Both minicircles and maxicircles were found to attach to these long DNA fibrils. The nature of the long DNA fibrils is not clear, but they may be important in providing a framework for the network structure and a support for the replication of minicircles and maxicircles.

show abstract

Isolation and characterization of kinetoplast DNA from Leishmania tarentolae

Cited by 100 publications

References 51 publications

Overproduction of a bifunctional thymidylate synthetase-dihydrofolate reductase and DNA amplification in methotrexate-resistant Leishmania tropica.

Overproduction of a bifunctional thymidylate synthetase-dihydrofolate reductase and DNA amplification in methotrexate-resistant Leishmania tropica.

The Structure of Kinetoplast DNA

Selection for arsenite resistance causes reversible changes in minicircle composition and kinetoplast organization in Leishmania mexicana.

Contact Info

Product

Resources

About