Article (Scientific journals)
High-efficiency biolistic transformation of Chlamydomonas mitochondria can be used to insert mutations in complex I genes
Remacle, Claire; Cardol, Pierre; Coosemans, Nadine et al.
2006In Proceedings of the National Academy of Sciences of the United States of America, 103 (12), p. 4771-4776
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Keywords :
green alga; mitochondrial DNA mutagenesis; telomere; complex; assembly; respiratory-deficient mutant
Abstract :
[en] Mitochondrial transformation of Chlamydomonas reinhardtii has been optimized by using a particle-gun device and cloned mitochondrial DNA or PCR fragments. A respiratory-deficient strain lacking a 1.2-kb mitochondrial DNA region including the left telomere and part of the cob gene could be rescued as well as a double-frameshift mutant in the mitochondrial cox1 and nd1 genes. High transformation efficiency has been achieved (100-250 transformants per microgram of DNA), the best results being obtained with linearized plasmid DNA. Molecular analysis of the transformants suggests that the right telomere sequence can be copied to reconstruct the left telomere by recombination. In addition, both nondeleterious and deleterious mutations could be introduced. Myxothiazol-resistant transformants have been created by introducing a nucleotide substitution into the cob gene. Similarly, an in-frame deletion of 23 codons has been created in the nd4 mitochondrial gene of both the deleted and frameshift recipient strains. These 23 codons are believed to encode the first transmembrane segment of the ND4 protein. This Delta nd4 mutation causes a misassembly of complex 1, with the accumulation of a subcomplex that is 250-kDa smaller than the wild-type complex 1. The availability of efficient mitochondrial transformation in Chlamydomonas provides an invaluable tool for the study of mitochondrial biogenesis and, more specifically, for site-directed mutagenesis of mitochondrially encoded subunits of complex 1, of special interest because the yeast Saccharomyces cerevisiae, whose mitochondrial genome can be manipulated virtually at will, is lacking complex 1.
Disciplines :
Phytobiology (plant sciences, forestry, mycology...)
Author, co-author :
Remacle, Claire  ;  Université de Liège - ULiège > Département des sciences de la vie > Génétique
Cardol, Pierre  ;  Université de Liège - ULiège > Département des sciences de la vie > Biochimie végétale
Coosemans, Nadine ;  Université de Liège - ULiège > Département des sciences de la vie > DES en biotechnologie
Gaisne, Mauricette
Bonnefoy, Nathalie
Language :
English
Title :
High-efficiency biolistic transformation of Chlamydomonas mitochondria can be used to insert mutations in complex I genes
Publication date :
21 March 2006
Journal title :
Proceedings of the National Academy of Sciences of the United States of America
ISSN :
0027-8424
eISSN :
1091-6490
Publisher :
Natl Acad Sciences, Washington, United States - Washington
Volume :
103
Issue :
12
Pages :
4771-4776
Peer reviewed :
Peer Reviewed verified by ORBi
Available on ORBi :
since 28 February 2010

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