A new method to identify flanking sequence tags in <it>chlamydomonas</it> using 3’-RACE

• Main Authors: Meslet-Cladière Laurence, Vallon Olivier
• Format: Article
• Language: English
• Published: BMC 2012-06-01
• Series: Plant Methods
• Subjects: Chlamydomonas; Transformation; Mutant library; 3’ UTR; Antibiotic resistance
• Online Access: http://www.plantmethods.com/content/8/1/21

<p>Abstract</p> <p>Background</p> <p>The green alga <it>Chlamydomonas reinhardtii</it>, 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.</p> <p>Results</p> <p>Here, we present a novel method to identify FSTs in insertional mutants of <it>Chlamydomonas</it>. 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 <it>PSBS3</it> (possibly involved in non-photochemical quenching), the NimA-related protein kinase <it>CNK2</it>, the mono-dehydroascorbate reductase <it>MDAR1</it>, the phosphoglycerate mutase <it>PGM5</it> etc..</p> <p>Conclusion</p> <p>We propose that our 3’-RACE FST method can be used to build large scale FST libraries in <it>Chlamydomonas</it> and other transformable organisms.</p>