PCR-based assay for mating type and diploidy in <i>Chlamydomonas</i>

Zamora, Ivan; Feldman, Jessica L.; Marshall, Wallace F.

doi:10.2144/04374bm01

Cited by 32 publications

(20 citation statements)

References 6 publications

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“…smt7-1 mat3-5, smt14-1 mat3-5, smt15-1 mat3-5, and smt16-1 mat3-5 were then mated to a SMT mat3-4 MT1 strain and vegetative diploids were selected on TAP agar containing 12 mg/ml paromomycin and 50 mg/ml emetine. Vegetative diploids with the genotypes mat3-4/mat3-5, SMT7/smt7-1 mat3-4/mat3-5, SMT14/smt14-1 mat3-4/mat3-5, SMT15/smt15-1 mat3-4/mat3-5, and SMT16/ smt16-1 mat3-4/mat3-5 were tested by PCR to confirm the presence of both mating types (Zamora et al 2004) and of both wild-type and mutant alleles of the SMT gene.…”

Section: Methodsmentioning

confidence: 99%

A Suppressor Screen in Chlamydomonas Identifies Novel Components of the Retinoblastoma Tumor Suppressor Pathway

Fang

Umen

2008

Genetics

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The retinoblastoma (RB) protein is a eukaryotic tumor suppressor and negative cell-cycle regulator. Chlamydomonas reinhardtii cells that lack the RB homolog MAT3 show loss of size checkpoint control and deregulated cell-cycle progression leading to the production of tiny cells. We carried out an insertional mutagenesis screen to isolate bypass suppressors of mat3 (smt mutants) that reverted the mat3 cell-size defect. Previously we reported that the loci encoding Chlamydomonas homologs of E2F and DP were frequently disrupted in this screen, indicating that the architecture of the canonical RB pathway is conserved in Chlamydomonas with MAT3/RB acting as a negative regulator upstream of E2F/DP. Here, we describe four novel smt mutants that moderately suppressed the cell-size checkpoint and cell-cycle phenotypes of mat3. As single mutants, three of the smt strains displayed no obvious phenotypes, and one had a slightly small phenotype. Strikingly, several smt double-mutant combinations synergized to cause enhanced suppression of mat3 and even to cause a large-cell phenotype that is comparable to that caused by loss of DP1. Molecular characterization of one smt mutant revealed that suppression is due to a defect in a gene encoding a putative small ubiquitin-like modifier (SUMO) peptidase. Our results reveal a complex genetic network that lies downstream of MAT3/RB and implicate protein sumoylation as an important step for cell-cycle progression in cells that are missing MAT3/RB. T HE retinoblastoma (RB) protein is a tumor suppressor and negative cell-cycle regulator that is conserved in animals, plants, green algae, and other eukaryotic lineages, but has been lost from yeasts and other fungi. In the past two decades intensive efforts have been made to understand how RB regulates cellcycle progression, cell proliferation, differentiation, and development.The canonical RB pathway involves the cell-cycleregulated interaction of RB or its homologs (also called pocket proteins) with a heterodimeric transcription factor composed of E2F and DP subunits. The RB-associated E2F/DP protein complex represses transcription of cellcycle genes, and this repression is released by removal of RB via phosphorylation. Subsequently, E2F/DPdependent transcription of cell-cycle genes allows S-phase entry and cell-cycle progression (Weinberg 1995;Harbour and Dean 2000;Knudsen and Knudsen 2006).Genetic screens in the fruit fly Drosophila melanogaster and in the roundworm Caenorhabditis elegans have led to new insights into the RB pathway, including the identification of functions that are cell-cycle independent (Lu and

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Section: Methodsmentioning

confidence: 99%

A Suppressor Screen in Chlamydomonas Identifies Novel Components of the Retinoblastoma Tumor Suppressor Pathway

Fang

Umen

2008

Genetics

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“…Random meiotic progeny isolated from among thousands of zygote colonies were individually grown in 96-well microtiter plates and pinned onto agar media plates supplemented with hygromycin or rapamycin. Mating type was scored using PCR genotyping (primers presented in Supplemental Table 1) (Zamora et al, 2004).…”

Section: Genetic Analysismentioning

confidence: 99%

“…Gametes of opposite mating type and containing different resistance markers were spread onto TAP agar containing 20 and 15 mg/mL zeocin shortly after mixing (1 to 2 h) and vegetative diploid colonies were picked and confirmed using PCR-based genotyping (Supplemental Table 1) (Zamora et al, 2004).…”

Section: Genetic Analysismentioning

confidence: 99%

Synergism between Inositol Polyphosphates and TOR Kinase Signaling in Nutrient Sensing, Growth Control, and Lipid Metabolism in Chlamydomonas

et al. 2016

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The networks that govern carbon metabolism and control intracellular carbon partitioning in photosynthetic cells are poorly understood. Target of Rapamycin (TOR) kinase is a conserved growth regulator that integrates nutrient signals and modulates cell growth in eukaryotes, though the TOR signaling pathway in plants and algae has yet to be completely elucidated. We screened the unicellular green alga Chlamydomonas reinhardtii using insertional mutagenesis to find mutants that conferred hypersensitivity to the TOR inhibitor rapamycin. We characterized one mutant, vip1-1, that is predicted to encode a conserved inositol hexakisphosphate kinase from the VIP family that pyrophosphorylates phytic acid (InsP 6 ) to produce the low abundance signaling molecules InsP 7 and InsP 8 . Unexpectedly, the rapamycin hypersensitive growth arrest of vip1-1 cells was dependent on the presence of external acetate, which normally has a growth-stimulatory effect on Chlamydomonas. vip1-1 mutants also constitutively overaccumulated triacylglycerols (TAGs) in a manner that was synergistic with other TAG inducing stimuli such as starvation. vip1-1 cells had reduced InsP 7 and InsP 8 , both of which are dynamically modulated in wild-type cells by TOR kinase activity and the presence of acetate. Our data uncover an interaction between the TOR kinase and inositol polyphosphate signaling systems that we propose governs carbon metabolism and intracellular pathways that lead to storage lipid accumulation.

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“…We therefore tested lines that had undergone LOH at the ACT2 locus for chromosome loss versus recombination, by analyzing the MT locus using PCR primers specific for the MT- and MT+ alleles [23]. As illustrated in Fig.…”

Section: Resultsmentioning

confidence: 99%

A mutation in the centriole-associated protein centrin causes genomic instability via increased chromosome loss in Chlamydomonas reinhardtii

Zamora

Marshall

2005

BMC Biol

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PCR-based assay for mating type and diploidy in Chlamydomonas

Cited by 32 publications

References 6 publications

A Suppressor Screen in Chlamydomonas Identifies Novel Components of the Retinoblastoma Tumor Suppressor Pathway

A Suppressor Screen in Chlamydomonas Identifies Novel Components of the Retinoblastoma Tumor Suppressor Pathway

Synergism between Inositol Polyphosphates and TOR Kinase Signaling in Nutrient Sensing, Growth Control, and Lipid Metabolism in Chlamydomonas

A mutation in the centriole-associated protein centrin causes genomic instability via increased chromosome loss in Chlamydomonas reinhardtii

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