Stable Amplified DNA in Drug-Resistant
            <i>Leishmania</i>
            Exists as Extrachromosomal Circles

Garvey, Edward P.; Santi, Daniel V.

doi:10.1126/science.3726545

Cited by 121 publications

(44 citation statements)

References 22 publications

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“…In contrast, a nonconservative model would require some alteration in the structure of the wild-type H-region chromosome, which was not observed. Similar data have been presented for the H-and R-region amplifications observed in certain lines of L. major (1,2,19). It is tempting to speculate that the amplification mechanism itself was conservative, perhaps occurring by a process involving localized overreplication followed by recombinations among the overreplicated DNAs.…”

Section: Discussionsupporting

confidence: 61%

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Amplified DNAs in laboratory stocks of Leishmania tarentolae: extrachromosomal circles structurally and functionally similar to the inverted-H-region amplification of methotrexate-resistant Leishmania major.

Petrillo-Peixoto¹,

Beverley²

1988

Mol. Cell. Biol.

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We describe the structure of amplified DNA that was discovered in two laboratory stocks of the protozoan parasite Leishmania tarentolae. Restriction mapping and molecular cloning revealed that a region of 42 kilobases was amplified 8-to 30-fold in these lines. Southern blot analyses of digested DNAs or chromosomes separated by pulsed-field electrophoresis showed that the amplified DNA corresponded to the H region, a locus defined originally by its amplification in methotrexate-resistant Leishmania major (S. M. Beverley, J. A. Coderre, D. V. Santi, and R. T. Schimke, Cell 38:431439, 1984). Similarities between the amplified DNA of the two species included (i) extensive cross-hybridization; (ii) approximate conservation of sequence order; (iii) extrachromosomal localization; (iv) an overall inverted, head-to-head configuration as a circular 140-kilobase tetrameric molecule; (v) two regions of DNA sequence rearrangement, each of which was closely associated with the two centers of the inverted repeats; (vi) association with methotrexate resistance; and (vii) phenotypically conservative amplification, in which the wild-type chromosomal arrangement was retained without apparent modification. Our data showed that amplified DNA mediating drug resistance arose in unselected L. tarentolae, although the pressures leading to apparently spontaneous amplification and maintenance of the H region are not known. The simple structure and limited extent of DNA amplified in these and other Leishmania lines suggests that the study of gene amplification in Leishmania spp. offers an attractive model system for the study of amplification in cultured mammalian cells and tumors. We also introduced a method for measuring the size of large circular DNAs, using gamma-irradiation to introduce limited double-strand breaks followed by sizing of the linear DNAs by pulsed-field electrophoresis.

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Section: Discussionsupporting

confidence: 61%

“…6B) exhibited the unusual mobility properties characteristic of supercoiled circular DNAs, such as pulse time-dependent mobility (relative to those of linear DNA size markers [1,19,22]). Using pulse times of 90 to 150 s, this DNA had an apparent molecular size of about 1,000 kb (Fig.…”

Section: Methodsmentioning

confidence: 99%

Amplified DNAs in laboratory stocks of Leishmania tarentolae: extrachromosomal circles structurally and functionally similar to the inverted-H-region amplification of methotrexate-resistant Leishmania major.

Petrillo-Peixoto¹,

Beverley²

1988

Mol. Cell. Biol.

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“…1, had apparent sizes in the range of 340 to 430 kb compared with those of the S. cerevisiae chromosomes and lambda multimers. This apparent change in sizes of bands with respect to the linear size standards and in response to pulse time variation would be expected if the DNA molecules were supercoiled circular plasmids (9,17,19). In contrast, Borrelia DNA bands in the range of 150 to 200 kb in Fig.…”

Section: Resultsmentioning

confidence: 87%

Conversion of a linear to a circular plasmid in the relapsing fever agent Borrelia hermsii

et al. 1996

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Spirochetes of the genusSpirochetes are a distinct phylum of eubacteria (41, 55). Known pathogenic members of this division are in the genera Treponema, Borrelia, and Leptospira. The genus Borrelia comprises microorganisms that are transmitted to vertebrates by hematophagous arthropods. Included in this genus are B. hermsii and B. turicatae, agents of relapsing fever in North America; B. burgdorferi sensu lato, the cause of Lyme disease; B. anserina, the agent of avian spirochetosis; and B. coriaceae, the putative agent of epidemic bovine abortion (5).The genomes of Borrelia spp., like those of many other bacteria, are composed of a chromosome and plasmids. What distinguishes the Borrelia genome from that of most other bacteria is its largely linear structure. The chromosome of B. burgdorferi is a linear duplex DNA molecule about 1 Mb in length (6,13,14,16,17). Linear plasmids ranging in size from 5 to 55 kb are present in B. burgdorferi and B. hermsii (2, 32, 43, 54); These linear plasmids carry genes for outer membrane lipoproteins, including OspA, OspB, and OspD in B. burgdorferi (3, 4) and the serotype-specifying Vmp proteins of B. hermsii (31, 43).Borrelia spp. also have circular DNA molecules, the form of extrachromosomal DNA in most bacteria (30, 51). Although these constitute only a small fraction of the Borrelia genome, circular plasmids carry what appear to be critical genes. In B. burgdorferi, these include ospC, which codes for an outer surface protein (35,46), and homologs of guaA and guaB, which code for enzymes involved in de novo purine biosynthesis (36).The structure of linear plasmids in B. burgdorferi has been the subject of detailed examination. Electron microscopic examination of the 49-kb linear plasmid of this species showed that the two strands of the duplex DNA are covalently closed at each end (4). The telomeres of the 16-and 49-kb plasmids of B. burgdorferi have been cloned, and sequence analysis of the telomeres revealed that there are short inverted repeats at their termini and that they have terminal hairpin loops (25, 28). A similar telomeric sequence was found on linear plasmids of B. hermsii, suggesting that other linear plasmids of this genus have similar structure (31). The telomeres of linear plasmids in Borrelia spp. have structural and sequence homology to those of some linear eukaryotic DNA viruses, specifically poxviruses and African swine fever virus (25).B. hermsii and B. burgdorferi have several genome equivalents arrayed along the lengths of the wavy filamentous cells (32). Linear and circular plasmids have a copy number of approximately one to two per chromosome in both B. burgdorferi and B. hermsii (14,26,32). This arrangement suggests a tight coupling of plasmid and chromosome replication and segregation in borrelias.To further understand the genetic organization of Borrelia spp., we examined the genomes of B. hermsii, B. burgdorferi, B. turicatae, and B. anserina in more detail. In the course of this characterization, we found linear double-stranded DNA molecules w...

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“…These experiments indicated that megabase-sized B. burgdorferi DNA was not a supercoiled circle. Although evidence to date suggests that open circular DNA migrates through a pulsed-field gel system, such as TAFE, very slowly if at all (19,21), we addressed the possibility that megabase-sized DNA of B. burgdorferi was an open circle by using twodimensional agarose gel electrophoresis. Open circular phage A DNA served as a control; it along with linear phage A DNA were driven a short distance into the agarose gel with a constant electrical field of low voltage.…”

Section: Methodsmentioning

confidence: 99%

Megabase-sized linear DNA in the bacterium Borrelia burgdorferi, the Lyme disease agent

Ferdows¹,

Barbour²

1989

Trends in Genetics

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Stable Amplified DNA in Drug-Resistant Leishmania Exists as Extrachromosomal Circles

Cited by 121 publications

References 22 publications

Amplified DNAs in laboratory stocks of Leishmania tarentolae: extrachromosomal circles structurally and functionally similar to the inverted-H-region amplification of methotrexate-resistant Leishmania major.

Amplified DNAs in laboratory stocks of Leishmania tarentolae: extrachromosomal circles structurally and functionally similar to the inverted-H-region amplification of methotrexate-resistant Leishmania major.

Conversion of a linear to a circular plasmid in the relapsing fever agent Borrelia hermsii

Megabase-sized linear DNA in the bacterium Borrelia burgdorferi, the Lyme disease agent

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