Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT01586 (G418)
 2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have demonstrated previously that Leishmania enriettii contains the enzymatic machinery to mediate efficient interplasmidic homologous recombination. In this report we show that a sequence insertion targeting vector, pALT-Neo-Tub, can be inserted into the genome of L. enriettii by homologous recombination between alpha-tubulin sequences found in the plasmid and their homologs in the genome. pALT-Neo-Tub, a pBluescript-derived vector containing the neor gene flanked by the alpha-tubulin intergenic and alpha-tubulin coding sequences, was used to transfect cells to G418 resistance. Analysis of the DNA from the drug resistant clones indicates that all of the insertion events are restricted to the alpha-tubulin gene repeats. As little as 200 base pairs of sequence homology between the plasmid and the genome is required for integration. Nonhomologous recombination events are not detected. These results indicate that exogenous DNA sequences can be integrated into the L. enriettii genome provided that they are flanked by homologous DNA sequences.
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PMID:A sequence insertion targeting vector for Leishmania enriettii. 153 57

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.
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PMID:Stable transformation of Leptomonas seymouri by circular extrachromosomal elements. 171 88

We have used derivatives of the recently developed stable transfection vector pALT-Neo to formally demonstrate that Leishmania enriettii contains the enzymatic machinery necessary for homologous recombination. This observation has implications for gene regulation, gene amplification, genetic diversity, and the maintenance of tandemly repeated gene families in the Leishmania genome as well as in closely related organisms, including Trypanosoma brucei. Two plasmids containing nonoverlapping deletions of the chloramphenicol acetyltransferase (CAT) gene, as well as the neomycin-resistance gene, were cotransfected into L. enriettii. Analysis of the DNA from these cells by Southern blotting and plasmid rescue revealed that a full-length or doubly deleted CAT gene could be reconstructed by homologous crossing-over and/or gene conversion between the two deletion plasmids. Additionally, parasites cotransfected with pALT-Neo and pALT-CAT-S, a plasmid containing two copies of the chimeric alpha-tubulin-CAT gene, resulted in G418-resistant parasites expressing high levels of CAT activity. The structure of the DNA within these cells, as shown by Southern blot analysis and the polymerase chain reaction, is that which would be expected from a homologous exchange event occurring between the two plasmids.
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PMID:Homologous recombination in Leishmania enriettii. 199 78