Stable transformation of Leptomonas seymouri by circular extrachromosomal elements.
To define the cis-acting sequences necessary for gene expression and DNA replication in trypanosomatids, we have developed a selectable vector that can be grown in Escherichia coli and maintained stably in the insect trypanosomatid Leptomonas seymouri. The vector is relatively small (6 kilobase pairs) and contains a portion of the L. seymouri alpha-tubulin gene positioned in-frame with a truncated neomycin phosphotransferase gene that confers resistance to the aminoglycoside G418. This construct is maintained in cells as a high-copy-number circular extrachromosomal element containing several head-to-tail copies of the transforming plasmid. In L. seymouri, alpha-tubulin-neomycin phosphotransferase fusion RNAs are polyadenylylated and possess a trans-spliced mini-exon. Additional DNA sequences can be inserted into the vector, propagated, and expressed in transformed cells.
Bellofatto
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Laboratory of Molecular Parasitology, Rockefeller University, New York, NY 10021.
Torres-Muñoz
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Cross
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D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012326:RNA Splicing; D012333:RNA, Messenger; D014158:Transcription, Genetic; D014162:Transfection; D014170:Transformation, Genetic; D014351:Trypanosomatina / Q000235:genetics*
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA