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First published online January 21, 2005; 10.1104/pp.104.050914

Plant Physiology 137:522-533 (2005)
© 2005 American Society of Plant Biologists

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Functional Genomics of the Regulation of the Nitrate Assimilation Pathway in Chlamydomonas1

David González-Ballester, Amaury de Montaigu, José Javier Higuera, Aurora Galván and Emilio Fernández*

Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Cordoba, Campus de Rabanales, 14071 Cordoba, Spain

The existence of mutants at specific steps in a pathway is a valuable tool of functional genomics in an organism. Heterologous integration occurring during transformation with a selectable marker in Chlamydomonas (Chlamydomonas reinhardtii) has been used to generate an ordered mutant library. A strain, having a chimeric construct (pNia1::arylsulfatase gene) as a sensor of the Nia1 gene promoter activity, was transformed with a plasmid bearing the paramomycin resistance AphVIII gene to generate insertional mutants defective at regulatory steps of the nitrate assimilation pathway. Twenty-two thousand transformants were obtained and maintained in pools of 96 for further use. The mutant library was screened for the following phenotypes: insensitivity to the negative signal of ammonium, insensitivity to the positive signal of nitrate, overexpression in nitrate, and inability to use nitrate. Analyses of mutants showed that (1) the number or integrated copies of the gene marker is close to 1; (2) the probability of cloning the DNA region at the marker insertion site is high (76%); (3) insertions occur randomly; and (4) integrations at different positions and orientations of the same genomic region appeared in at least three cases. Some of the mutants analyzed were found to be affected at putative new genes related to regulatory functions, such as guanylate cyclase, protein kinase, peptidyl-prolyl isomerase, or DNA binding. The Chlamydomonas mutant library constructed would also be valuable to identify any other gene with a screenable phenotype.

1 This work was supported by Ministerio de Ciencia y Tecnología, Spain (MCYT grant no. BMC2002–03325); European Commission (RTN grant HPRN–CT–2002–00247); and Junta de Andalucía, Spain (PAI grant CVI–0128).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.050914.

* Corresponding author; e-mail bb1feree{at}uco.es; fax 34–957–218591.

Received July 30, 2004; returned for revision October 13, 2004; accepted October 14, 2004.




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