Article

Generation of an oligonucleotide array for analysis of gene expression in Chlamydomonas reinhardtii

Carnegie Institute, Stanford, California, United States
Current Genetics (Impact Factor: 2.68). 03/2006; 49(2):106-24. DOI: 10.1007/s00294-005-0041-2
Source: PubMed

ABSTRACT

The availability of genome sequences makes it possible to develop microarrays that can be used for profiling gene expression over developmental time, as organisms respond to environmental challenges, and for comparison between wild-type and mutant strains under various conditions. The desired characteristics of microarrays (intense signals, hybridization specificity and extensive coverage of the transcriptome) were not fully met by the previous Chlamydomonas reinhardtii microarray: probes derived from cDNA sequences (approximately 300 bp) were prone to some nonspecific cross-hybridization and coverage of the transcriptome was only approximately 20%. The near completion of the C. reinhardtii nuclear genome sequence and the availability of extensive cDNA information have made it feasible to improve upon these aspects. After developing a protocol for selecting a high-quality unigene set representing all known expressed sequences, oligonucleotides were designed and a microarray with approximately 10,000 unique array elements (approximately 70 bp) covering 87% of the known transcriptome was developed. This microarray will enable researchers to generate a global view of gene expression in C. reinhardtii. Furthermore, the detailed description of the protocol for selecting a unigene set and the design of oligonucleotides may be of interest for laboratories interested in developing microarrays for organisms whose genome sequences are not yet completed (but are nearing completion).

    • "It has been proposed that MTCs are the evolutionary origins of mammalian caspases (Koonin & Aravind, 2002), but there is a long-standing debate on whether they fill the same functional role during cell death or also function in other cellular processes, as some forms of PCD are independent of MTCs (Vercammen et al., 2007; Carmona-Gutierrez et al., 2010). The green alga Chlamydomonas reinhardtii (hereafter – Chlamydomonas) often serves as a model organism to study various cellular aspects; many mutants are available and various genetic tools have been developed (Eberhard et al., 2006; Merchant et al., 2007; Fischer et al., 2012). We have shown previously that exposure to H 2 O 2 treatment of elder cultures of both Chlamydomonas and the dinoflagellate Peridinium gatunense, close to the inflection point in their growth curve, results in the typical hallmarks of apoptosis, including an elevated expression and activity of MTCs (Murik & Kaplan, 2009). "

    No preview · Article · Aug 2014 · New Phytologist
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    • "It has been proposed that MTCs are the evolutionary origins of mammalian caspases (Koonin & Aravind, 2002), but there is a long-standing debate on whether they fill the same functional role during cell death or also function in other cellular processes, as some forms of PCD are independent of MTCs (Vercammen et al., 2007; Carmona-Gutierrez et al., 2010). The green alga Chlamydomonas reinhardtii (hereafter – Chlamydomonas) often serves as a model organism to study various cellular aspects; many mutants are available and various genetic tools have been developed (Eberhard et al., 2006; Merchant et al., 2007; Fischer et al., 2012). We have shown previously that exposure to H 2 O 2 treatment of elder cultures of both Chlamydomonas and the dinoflagellate Peridinium gatunense, close to the inflection point in their growth curve, results in the typical hallmarks of apoptosis, including an elevated expression and activity of MTCs (Murik & Kaplan, 2009). "
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    ABSTRACT: Chlamydomonas reinhardtii tolerates relatively high H2 O2 levels that induce an array of antioxidant activities. However, rather than rendering the cells more resistant to oxidative stress, the cells become far more sensitive to an additional H2 O2 dose. If H2 O2 is provided 1.5-9 h after an initial dose, it induces programmed cell death (PCD) in the wild-type, but not in the dum1 mutant impaired in the mitochondrial respiratory complex III. This mutant does not exhibit a secondary oxidative burst 4-5 h after the inducing H2 O2 , nor does it activate metacaspase-1 after the second H2 O2 treatment. The intracellular dehydroascorbate level, a product of ascorbate peroxidase, increases under conditions leading to PCD. The addition of dehydroascorbate induces PCD in the wild-type and dum1 cultures, but higher levels are required in dum1 cells, where it is metabolized faster. The application of dehydroascorbate induces the expression of metacaspase-2, which is much stronger than the expression of metacaspase-1. The presence or absence of oxidative stress, in addition to the rise in internal dehydroascorbate, may determine which metacaspase is activated during Chlamydomonas PCD. Cell death is strongly affected by the timing of H2 O2 or dehydroascorbate admission to synchronously grown cultures, suggesting that the cell cycle phase may distinguish cells that perish from those that do not.
    Full-text · Article · Dec 2013 · New Phytologist
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    • "Red dots represent up-regulated genes and green dots down-regulated genes. with a different first-generation microarray platform (Eberhard et al., 2006). It should be noted that the microarray platform used in our study included almost 4000 new gene models including a large number of unknown gene models (Toepel et al., 2011). "
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    ABSTRACT: Hydrogen production with Chlamydomonas reinhardtii induced by sulphur starvation is a multiphase process while the cell internal metabolism is completely remodelled. The first cellular response is characterized by induction of genes with regulatory functions, followed by a total remodelling of the metabolism to provide reduction equivalents for cellular processes. We were able to characterize all major processes that provide energy and reduction equivalents during hydrogen production. Furthermore, C. reinhardtii showed a strong transcript increase for gene models responsible for stress response and detoxification of oxygen radicals. Finally, we were able to determine potential bottlenecks and target genes for manipulation to increase hydrogen production or to prolong the hydrogen production phase. The investigation of transcriptomic changes during the time course of hydrogen production in C. reinhardtii with microarrays and RNA-seq revealed new insights into the regulation and remodelling of the cell internal metabolism. Both methods showed a good correlation. The microarray platform can be used as a reliable standard tool for routine gene expression analysis. RNA-seq additionally allowed a detailed time-dependent study of gene expression and determination of new genes involved in the hydrogen production process.
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