Use of the ARG7 gene as an insertional mutagen to clone PHON 24,  a gene required for derepressible neutral phosphatase activity  in Chlamydomonas reinhardtii

Adam, M; Loppes, Roland

doi:10.1007/s004380050714

Cited by 18 publications

(21 citation statements)

References 47 publications

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“…Genomic DNA was digested with the PstI restriction enzyme and probed using the 130-bp StuI-SphI fragment from the Arg7 gene. This probe contains little redundant genomic sequence and is specific for Arg7 (Debuchy et al, 1989;Adam and Loppes, 1998). As shown in Figure 3, the sta7-10 mutant exhibits one additional site of hybridization in addition to the nonfunctional wild-type copy of the arg7 gene.…”

Section: Mutant Isolation and Characterizationmentioning

confidence: 99%

“…Screening DNA insertional mutagenesis libraries in C. reinhardtii has become a popular strategy for identifying important genes involved in specific cellular pathways and processes (Debuchy et al, 1989;Rochaix, 1995;Tam and Lefebvre, 1995;Niyogi et al, 1997;Adam and Loppes, 1998;Davies et al, 1999;Moseley et al, 2000; Van et al, 2001;Dame et al, 2002;Polle et al, 2003). Our screening efforts resulted in the identification of several mutants that were compromised in their ability to photoproduce H 2 .…”

Section: Introductionmentioning

confidence: 99%

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Hydrogen Photoproduction Is Attenuated by Disruption of an Isoamylase Gene in Chlamydomonas reinhardtii

et al. 2004

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DNA insertional transformants of Chlamydomonas reinhardtii were screened chemochromically for attenuated H 2 production. One mutant, displaying low H 2 gas photoproduction, has a nonfunctional copy of a gene that shows high homology to the family of isoamylase genes found in several photosynthetic organisms. DNA gel blotting and gene complementation were used to link this isoamylase gene to previously characterized nontagged sta7 mutants. This mutant is therefore denoted sta7-10. In C. reinhardtii, the STA7 isoamylase gene is important for the accumulation of crystalline starch, and the sta7-10 mutant reported here contains <3% of the glucose found in insoluble starch when compared with wild-type control cells. Hydrogen photoproduction rates, induced after several hours of dark, anaerobic treatment, are attenuated in sta7 mutants. RNA gel blot analysis indicates that the mRNA transcripts for both the HydA1 and HydA2 [Fe]-hydrogenase genes are expressed in the sta7-10 mutant at greater than wild-type levels 0.5 h after anaerobic induction. However, after 1.5 h, transcript levels of both HydA1 and HydA2 begin to decline rapidly and reach nearly undetectable levels after 7 h. In wildtype cells, the hydrogenase transcripts accumulate more slowly, reach a plateau after 4 h of anaerobic treatment, and maintain the same level of expression for >7 h under anaerobic incubation. Complementation of mutant cells with genomic DNA corresponding to the STA7 gene restores both the starch accumulation and H 2 production phenotypes. The results indicate that STA7 and starch metabolism play an important role in C. reinhardtii H 2 photoproduction. Moreover, the results indicate that mere anaerobiosis is not sufficient to maintain hydrogenase gene expression without the underlying physiology, an important aspect of which is starch metabolism.

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Section: Mutant Isolation and Characterizationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Hydrogen Photoproduction Is Attenuated by Disruption of an Isoamylase Gene in Chlamydomonas reinhardtii

et al. 2004

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“…Transformations of strain TerBD20 with plasmid pASL (Adam and Loppez, 1998) were performed following method described by Kindle (1990).…”

Section: Insertional Mutagenesismentioning

confidence: 99%

STA11, a Chlamydomonas reinhardtii Locus Required for Normal Starch Granule Biogenesis, Encodes Disproportionating Enzyme. Further Evidence for a Function of α-1,4 Glucanotransferases during Starch Granule Biosynthesis in Green Algae

et al. 2003

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In Chlamydomonas reinhardtii, the presence of a defective STA11 locus results in significantly reduced granular starch deposition displaying major modifications in shape and structure. This defect simultaneously leads to the accumulation of linear malto-oligosaccharides (MOS). The mutants of STA11 were showed to lack d-enzyme, a plant ␣-1,4 glucanotransferase analogous to the Escherichia coli amylomaltase. We have cloned and characterized both the cDNA and gDNA corresponding to the C. reinhardtii d-enzyme. We now report allele-specific modifications of the d-enzyme gene in the mutants of STA11. These allele-specific modifications cosegregate with the corresponding sta11 mutations, thereby demonstrating that STA11 encodes d-enzyme. MOS production and starch accumulation were investigated during day and night cycles in wild-type and mutant C. reinhardtii cells. We demonstrate that in the algae MOS are produced during starch biosynthesis and degraded during the phases of net polysaccharide catabolism.Until recently plant starch was believed to be synthesized from ADP-Glc solely through a combination of starch synthases and branching enzymes. However the finding of low-starch or starchless mutants defective for a particular form of debranching enzyme in four different plant systems established that other enzymes of starch metabolism are equally important in ensuring normal starch granule biogenesis (James et al., 1995;Mouille et al., 1996;Nakamura et al., 1996;Zeeman et al., 1998). This came as a surprise because debranching enzymes were initially thought to be enzymes involved solely in starch breakdown. Mutants of the corresponding activities in yeast (Teste et al., 2000) or Escherichia coli (I. Kinderf, Z. Li, M.S. Samuel, B. Koshar-Hashezmi, S. Ball, L. Rampling, and M. Morell, unpublished data) are clearly glycogen over-producers, confirming the initial suspicion. Although the detailed interpretation of the results obtained with the plant mutants vary somewhat, there is now a general agreement that isoamylases, the particular form of debranching enzyme affected in these studies, are enzymes required during starch biosynthesis exclusively. It has been comforting to realize that all mutants affected in starch metabolism behaved in a similar fashion in plants as different as Chlamydomonas reinhardtii, Arabidopsis, pea (Pisum sativum), maize (Zea mays), or rice (Oryza sativa). Some discrepancies in expressivity of mutant phenotypes could be easily explained most of the time by subtle differences in the pathways. For instance, the presence of extraplastidial and plastidial ADP-Glc pyrophosphorylases in cereals easily explains why mutants of cereals lacking the major enzyme form displayed reduced expressivity in their starch accumulation phenotype (for review, see Kossmann and Lloyd, 2000). The starch accumulation phenotypes of the Arabidopsis or C. reinhardtii mutants defective for the catalytic subunit of their sole plastidial ADP-Glc pyrophosphorylase were much more severe (Lin et al., 1988;Zabawinski et al., 2001...

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“…Transformation was performed using 0.6 Wg BamHI-linearized pASL (pBCKS +Chlamydomonas ARG7 gene [10]) as previously described [11]. Crosses, maturation of zygotes and random analysis of meiotic products were carried out according to published procedures [9].…”

Section: Reinhardtii Strains Culture Transformation and Genetic mentioning

confidence: 99%

“…About 2U10 8 cells were resuspended in 700 Wl of CTAB bu¡er (2% (w/v) cetyltrimethylammonium bromide, 100 mM Tris^HCl pH 8.0, 1.4 M NaCl, 20 mM EDTA pH 8.0, 2% (v/v) L-mercaptoethanol) and incubated at 65³C for 1 h. After two phenol^chloroform extractions, DNA was precipitated with 0.7 volume isopropanol, resuspended in TE and treated with ribonuclease A (20 Wg ml 31 ). Southern blot analysis was carried out as previously described [10] using the 130-bp StuI^SphI fragment from exon 11 of the Chlamydomonas ARG7 gene as a probe.…”

Section: Dna Preparation and Hybridization Proceduresmentioning

confidence: 99%

Pleiotropic mutants hypersensitive to heavy metals and to oxidative stress in Chlamydomonas reinhardtii

Hanikenne

2001

FEMS Microbiology Letters

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Insertional mutagenesis was used in Chlamydomonas reinhardtii to isolate original mutants hypersensitive to multiple drugs and physical agents. Out of 5200 transformants analyzed, 13 mutants belonging to seven phenotypic classes were isolated. Five were exclusively sensitive to cadmium and represented two loci. The other mutants were pleiotropic and presented a cross sensitivity to several (2^6) of the following agents: cadmium, copper, lead, paraquat, hydrogen peroxide, UVC and light. In all mutants analyzed, the hypersensitive phenotype was most probably due to a single mutational event. The sensitivity of several pleiotropic mutants to a broad range of physical and chemical agents suggests that the disrupted genes are involved in multiple stress responses. ß

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Use of the ARG7 gene as an insertional mutagen to clone PHON 24, a gene required for derepressible neutral phosphatase activity in Chlamydomonas reinhardtii

Cited by 18 publications

References 47 publications

Hydrogen Photoproduction Is Attenuated by Disruption of an Isoamylase Gene in Chlamydomonas reinhardtii

Hydrogen Photoproduction Is Attenuated by Disruption of an Isoamylase Gene in Chlamydomonas reinhardtii

STA11, a Chlamydomonas reinhardtii Locus Required for Normal Starch Granule Biogenesis, Encodes Disproportionating Enzyme. Further Evidence for a Function of α-1,4 Glucanotransferases during Starch Granule Biosynthesis in Green Algae

Pleiotropic mutants hypersensitive to heavy metals and to oxidative stress in Chlamydomonas reinhardtii

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