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

Journal Article · Thu Dec 01 00:00:00 EST 1988 · Molecular and Cellular Biology; (USA)

DOI:https://doi.org/10.1128/MCB.8.12.5188· OSTI ID:5401311

Petrillo-Peixoto, M L; Beverley, S M ^[1]

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1. Harvard Medical School, Boston, MA (USA)

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. 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.

• OSTI ID: 5401311
• Journal Information: Molecular and Cellular Biology; (USA), Vol. 8:12; ISSN 0270-7306
• Country of Publication: United States
• Language: English

Related Subjects

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
DNA
MOLECULAR STRUCTURE
PROTOZOA
GENE AMPLIFICATION
STRAND BREAKS
RADIOINDUCTION
CHROMOSOMES
DNA HYBRIDIZATION
DNA-CLONING
ELECTROPHORESIS
GAMMA RADIATION
GENETIC MAPPING
METHOTREXATE
PHENOTYPE
ANIMALS
ANTIMETABOLITES
CLONING
DRUGS
ELECTROMAGNETIC RADIATION
HYBRIDIZATION
INVERTEBRATES
IONIZING RADIATIONS
MAPPING
MICROORGANISMS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
RADIATIONS
560130* - Radiation Effects on Microorganisms