Creation of Null/+ mutants of the alpha-tubulin gene in Leishmania enriettii by gene cluster deletion.
Tubulins are abundant structural proteins in the Leishmania parasite, and tubulin genes are examples of highly expressed genes, which are present in multiple copies arranged in tandem repeats. Functional analysis of such multicopy genes using genetic manipulation has not yet been possible. Here we describe a method for creating deletions of alpha-tubulin gene clusters by targeted gene replacement and report the isolation of null/+ mutants deleted for either of the two allelic tubulin clusters. We also report null/null mutants in which both clusters have been deleted from their chromosomal loci and in which alpha-tubulin genes are present as episomal elements. Characterization of tubulin mRNA expression in these mutants indicated a posttranscriptional up-regulation of alpha-tubulin mRNA stability in null/+ mutants which contained only one-third the normal number of alpha-tubulin genes. A parallel increase in beta-tubulin mRNA levels indicated coordinate regulation of tubulin mRNA in Leishmania.
Curotto de Lafaille
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MA
Department of Tropical Public Health, Harvard School of Public Health, Boston, Massachusetts 02115.
Wirth
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D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D017353:Gene Deletion; D015870:Gene Expression; D005822:Genetic Vectors; D007438:Introns; D007894:Leishmania mexicana / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D014404:Tubulin / Q000235:genetics*
D016054:DNA, Protozoan; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D014404:Tubulin