A minus-gamete-specific gene in fusion-defective Chlamydomonas mutants and analysis of biosynthetic pathways upregulated during gametogenesis
Item
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Title
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A minus-gamete-specific gene in fusion-defective Chlamydomonas mutants and analysis of biosynthetic pathways upregulated during gametogenesis
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Identifier
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d_2009_2013:4ec5620ab357:11636
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identifier
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12229
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Creator
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Brogun, Dmitry Y.,
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Contributor
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Charlene L. Forest
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Date
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2013
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Language
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English
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Publisher
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City University of New York.
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Subject
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Molecular biology | Systematic biology | biosynthesis | Chlamydomonas reinhardtii | fusion | gamete | isoprenoid | phylogenetics
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Abstract
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To gain insight into the mechanism of gamete fusion during fertilization, it is crucial to identify molecular, metabolic and genetic factors required for this process. Gamete fusion in C. reinhardtii cells proceeds via four genetically defined stages: 1) flagella recognition 2) signaling 3) mating structure adhesion and 4) fusion with subsequent zygote formation. During this study we used insertional and temperature sensitive conditional mutants that do not proceed to stage 4, but can agglutinate and adhere to each other via their mating structure. A homolog of the sex-restricted HAP2/GCS1 gene has been shown to prevent gamete fusion in C. reinhardtii. A SiteFinding-PCR search was conducted on the fusion-defective Chlamydomonas insertional mutants that could not be complemented with the wild type copy of the HAP2/GCS1 gene. We confirmed that mutant clone 5 had an insert in a copia-family retrotransposon on chromosome 13. We collaborated to discover that a gene, located 3' to the retrotransposon is a minus-gamete specific gene (MGS). We hypothesized that MGS might be a second gene required for gamete fusion. Our main objective was to identify whether there is a defect in the DNA sequence in MGS in any of our fusion-defective mutants. We performed a chromosome walk on coding, promoter and 5' upstream and 3' downstream regulatory regions (UTR) of MGS via PCR. PCR products then where sequenced and aligned. We used qRT-PCR to determine MGS expression levels in the control and fusion defective mutants. Analysis of the sequencing and expressional results showed no defect in the MGS gene.;For the systematics study we used comparative genomic and phylogenetic approaches enabling us to study metabolic pathways that are upregulated in gametes of Chlamydomonas. Congruent experimental results show that the nuclear-encoded and chloroplast localized MEP pathway leading to the biosynthesis of the isoprenoid precursor molecules is upregulated in Chlamydomonas gametes. It is expected, that the results from these studies will provide further insights into regulatory mechanisms occurring during gametogenesis, some of which might be necessary for gamete fusion in algae as well as in higher eukaryotic organisms.
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Type
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dissertation
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Source
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2009_2013.csv
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degree
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Ph.D.
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Program
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Biology
