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Mol. Cell. Biol., Oct 1995, 5762-5769, Vol 15, No. 10
JA Nelson and PA Lefebvre
We have used homologous recombination to disrupt the nuclear gene NIT8 in
Chlamydomonas reinhardtii. This is the first report of targeted gene
disruption of an endogenous locus in C. reinhardtii and only the second for
a photosynthetic eukaryote. NIT8 encodes a protein necessary for nitrate
and nitrite assimilation by C. reinhardtii. A disruption vector was
constructed by placing the CRY1-1 selectable marker gene, which confers
emetine resistance, within the NIT8 coding region. nit8 mutants are unable
to grow on nitrate as their sole nitrogen source (Nit-) and are resistant
to killing by chlorate. One of 2,000 transformants obtained after selection
on emetine-chlorate medium contained a homologous insertion of five copies
of the disruption plasmid into the NIT8 gene, producing an
emetine-resistant, chlorate-resistant Nit- phenotype. The mutant phenotype
was rescued by the wild-type NIT8 gene upon transformation. Seven other
mutations at the nit8 locus, presumably resulting from homologous
recombination with the disruption plasmid, were identified but were shown
to be accompanied by deletions of the surrounding genomic region.
Copyright © 1995, American Society for Microbiology
Targeted disruption of the NIT8 gene in Chlamydomonas reinhardtii
Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108, USA.
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