Stable Transfection oftheHumanParasite Leishmania major Delineates a30-Kilobase Region Sufficient forExtrachromosomal Replication andExpression

synthase andabacterial origin ofreplication andselectable marker added. G418-resistant lines wereobtained athighefficiency byelectroporation ofpR-NEO(approaching 10-4 percell), while constructs bearing aninverted neogeneorlacking Leishmania sequences didnotconfer resistance. pR-NEOreplicated inL.majorandgaverisetocorrectly processed transcripts bearing the trans-spliced miniexon. Molecular karyotype analysis showedthatinsomelines pR-NEODNA exists exclusively asanextrachromosomal circle, a finding supported bytherescue ofintact pR-NEOafter transformation ofEscherichia coli. Thesedatagenetically localize allelements required incisforDNA replication, transcription, andtrans splicing totheLeishmania DNAcontained within pR-NEODNAandsignal theadventofstable transfection methodology foraddressing molecular phenomena intrypanosomatid parasites.