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PMID: 2304458 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression.

Molecular and cellular biology ·Vol. 10 ·No. 3 ·1990-03-00 ·Pages 1084-94

Kapler GM, Coburn CM, Beverley SM

Abstract

To delineate segments of the genome of the human protozoan parasite Leishmania major necessary for replication and expression, we developed a vector (pR-NEO) which can be reproducibly introduced into L. major. This DNA was derived from a 30-kilobase extrachromosomal amplified DNA bearing the dihydrofolate reductase-thymidylate synthase gene, with the coding region for neomycin phosphotransferase substituted for that of dihydrofolate reductase-thymidylate synthase and a bacterial origin of replication and selectable marker added. G418-resistant lines were obtained at high efficiency by electroporation of pR-NEO (approaching 10(-4) per cell), while constructs bearing an inverted neo gene or lacking Leishmania sequences did not confer resistance. pR-NEO replicated in L. major and gave rise to correctly processed transcripts bearing the trans-spliced miniexon. Molecular karyotype analysis showed that in some lines pR-NEO DNA exists exclusively as an extrachromosomal circle, a finding supported by the rescue of intact pR-NEO after transformation of Escherichia coli. These data genetically localize all elements required in cis for DNA replication, transcription, and trans splicing to the Leishmania DNA contained within pR-NEO DNA and signal the advent of stable transfection methodology for addressing molecular phenomena in trypanosomatid parasites.

MeSH Terms
Animals Base Sequence Blotting, Northern DNA Replication DNA, Recombinant Extrachromosomal Inheritance Genetic Vectors Leishmania tropica/genetics Molecular Sequence Data Transcription, Genetic Transfection
Chemicals
DNA, Recombinant
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Kapler G M
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Coburn C M
Beverley S M
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.10.3.1084-1094.1990
Published
1990-03-00
Pages
1084-94
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC360971
Subset
IM
Grants
NIGMS NIH HHS · 5T-32-GM07306 · United States
NIAID NIH HHS · AI-2903 · United States
NCRR NIH HHS · S07 RR 05381-27 · United States
External Links
PubMed source DOI: 10.1128/mcb.10.3.1084-1094.1990 PMC Full Text View journal details
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