The 63-kilobase circular amplicon of tunicamycin-resistant Leishmania amazonensis contains a functional N-acetylglucosamine-1-phosphate transferase gene that can be used as a dominant selectable marker in transfection.

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RESUMO

Tunicamycin (TM)-resistant Leishmania amazonensis has been found previously to contain amplified chromosomal DNA, existing exclusively as extrachromosomal circles of 63 kb. Fragments of this DNA cloned into plasmids were functionally analyzed by transfection of wild-type cells. A clone with a 15-kb fragment of the 63-kb circle was initially found to confer TM resistance. A library of the 15-kb fragment was then prepared and used in toto to transfect wild-type cells. The transfectants that emerged after selection were found to contain a plasmid with an insert of 4.6 kb. Evidence from deletion experiments suggests that this is the minimal transfection-effective fragment. Sequencing of the 4.6-kb DNA revealed 1.4-kb homolog of N-acetylglucosamine-1-phosphate transferase genes. The L. amazonensis gene is similar to those from two other sources in their deduced peptide sequence by 65 to 70% and in hydropathic characteristics. The L. amazonensis gene is amplified by more than 128-fold over the wild type and overproduces a major transcript of 2.4 kb in all transfectants. The endogenous copy of this gene was amplified by polymerase chain reaction from the wild type and cloned into pX-NEO, a Leishmania expression vector. Amplification of this plasmid in the transfectants by selection with G418 simultaneously made them resistant to TM. Evidence provided thus indicates that the 1.4-kb DNA is an N-acetylglucosamine-1-phosphate transferase gene whose amplification is responsible for TM resistance in Leishmania variants and transfectants.

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