ArticlePlant methods2012
A new method to identify flanking sequence tags in chlamydomonas using 3'-RACE.
Article in Plant methods, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 7 papers.
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Who cites it
7 citing papers in PubMed, 20 citations in OpenAlex.
- Application of transposon insertion site sequencing method in the exploration of gene function in microalgae.Frontiers in microbiology · 2023Review
- Article
- MAPINS, a Highly Efficient Detection Method That Identifies Insertional Mutations and Complex DNA Rearrangements.Plant physiology · 2018Article
- Identification of Insertion Site by RESDA-PCR inBio-protocol · 2018Article
- A robust protocol for efficient generation, and genomic characterization of insertional mutants ofPlant methods · 2017Article
- An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii.The Plant cell · 2016Article
- High-Throughput Genotyping of Green Algal Mutants Reveals Random Distribution of Mutagenic Insertion Sites and Endonucleolytic Cleavage of Transforming DNA.The Plant cell · 2014Article
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundThe green alga Chlamydomonas reinhardtii, although a premier model organism in biology, still lacks extensive insertion mutant libraries with well-identified Flanking Sequence Tags (FSTs). Rapid and efficient methods are needed for FST retrieval.
resultsHere, we present a novel method to identify FSTs in insertional mutants of Chlamydomonas. Transformants can be obtained with a resistance cassette lacking a 3' untranslated region (UTR), suggesting that the RNA that is produced from the resistance marker terminates in the flanking genome when it encounters a cleavage/polyadenylation signal. We have used a robust 3'-RACE method to specifically amplify such chimeric cDNAs. Out of 38 randomly chosen transformants, 27 (71%) yielded valid FSTs, of which 23 could be unambiguously mapped to the genome. Eighteen of the mutants lie within a predicted gene. All but two of the intragenic insertions occur in the sense orientation with respect to transcription, suggesting a bias against situations of convergent transcription. Among the 14 insertion sites tested by genomic PCR, 12 could be confirmed. Among these are insertions in genes coding for PSBS3 (possibly involved in non-photochemical quenching), the NimA-related protein kinase CNK2, the mono-dehydroascorbate reductase MDAR1, the phosphoglycerate mutase PGM5 etc..
conclusionWe propose that our 3'-RACE FST method can be used to build large scale FST libraries in Chlamydomonas and other transformable organisms.
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