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

Abstract

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.

Isolation and characterization of pigment deficient insertional mutants in the chlorophyte Chlamydomonas reinhardtii

“…The number of integrations was calculated using the delta Ct method [38] and specific primers of very similar efficiencies for AphVIII and Nia1 ( Table I). Efficiencies of the primers were previously determined [21]. We found that practically all of the mutants analyzed had a single copy of the tag DNA in their genomes.…”

“…In mutants B20 and M2 several attempts did not yield any amplification. This could mean that the insertion has taken place in a region of low accessibility or that silencing epigenetic phenomena that modify the structure of the marker gene are interfering with the alignment of primers during PCR amplifications as proposed by González-Ballester and coworkers [21]. In mutant D45, predicted region coincides with the region of the chromosome 8, where is located the promoter of the Hsp70A gene and within the region of chromosome 2 corresponding to the RbcS2 promoter, which are part of the same insertional cassette, indicating the possible insertion of several tandem copies of the marker gene, as was already predicted when determining the number of integrations.…”

“…The AphVIII cassette was chosen as selectable marker because its high level of expression has already been shown [23] and the selective antibiotic paromomycin lacks the mutagenic effect reported for bleomycin [14]. No more than 100 ng of DNA per transformation were used in mutagenesis experiments since this small quantity of DNA is enough to ensure a good number of transformants minimizing the integration of multiple copies of the marker DNA, as has been set up by González-Ballester and coworkers [21]. Single integration is desirable to establish an unequivocal relation between the observed phenotype and the disrupted gene.…”

“…The DNA was kept to the minimum in the transformation experiments and the number of copies of the tag DNA was checked by Real Time PCR using the singlecopy gene Nitrate Reductase 1 (Nia1) as reference. This method has been previously validated by comparison with southern blot analysis [21]. The number of integrations was calculated using the delta Ct method [38] and specific primers of very similar efficiencies for AphVIII and Nia1 ( Table I).…”

“…This approach has allowed isolation of mutants affected in different metabolic and physiological aspects [14] and has become a popular method for forward genetics studies because identification of the affected genomic region is generally easier than location of genomic lesions caused by traditional mutagenesis procedures based on chemical or physical agents. In C. reinhardtii, this technique has allowed the isolation of mutants affected in flagellar function [15], phototaxis [16], microtubules [17], carbon assimilation [18], H 2 production [19], photosynthesis [20], nitrogen [21] and sulphur metabolism [22].…”