Biotechnology
| Poster #522 | |
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| » | Introduction |
| » | Materials and Methods |
| » | Results and Discussion |
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Characterization of a Suppressor of Epigenetic
Gene Silencing in Chlamydomonas reinhardtiiH. Cerutti1, D. Scharf1, J. Kovar2, and J. Qiu2
1 University of Nebraska-Lincoln, Lincoln, NE 68588; 2 LI-COR Inc., Lincoln, NE 68504
Abstract
We have constructed a dominant selectable marker for nuclear transformation of the unicellular green alga Chlamydomonas reinhardtii. It is composed of the coding sequence of the eubacterial aadA gene (conferring spectinomycin resistance) under the control of the 5' and 3' regulatory regions of the Chlamydomonas RbcS2 gene (encoding the small subunit of Rubisco). This chimeric RbcS2:aadA:RbcS2 gene integrated stably into the nuclear genome but the spectinomycin resistance phenotype was unstable in many transformants. Clonal pedigree analyses and nuclear run-on assays indicated that expression of the RbcS2:aadA:RbcS2 transgene was being repressed by an epigenetic mechanism(s).
We have isolated several tagged mutants, generated by random insertion of transforming plasmid DNA, that stably reactivate expression of the silenced RbcS2:aadA:RbcS2 transgene. One of the disrupted genes has now been cloned and encodes a putative RNA helicase of the DEAH-box superfamily with homology to Prp16, an essential splicing factor in Saccharomyces cerevisiae. The Chlamydomonas mutant also reactivates expression of a retrotransposon (TOC1) and shows higher levels of aberrantly processed (e.g., shorter, improperly spliced, etc.) RbcS2:aadA:RbcS2 transcripts. We hypothesize that the putative RNA helicase may be involved in the degradation of aberrant mRNAs and/or in controlling the fidelity of splicing. However, it does not appear to be an essential splicing factor since the processing of two endogenous genes, RbcS2 and TubA (encoding a tubulin), is not affected in the mutant strain. To understand further the molecular role of the cloned RNA helicase, we are trying to identify transcripts that are differentially expressed in the mutant vs. wild type strains using a PCR-based method which allows expression changes to be determined in an undirected survey of unknown genes.
