A new method to identify flanking sequence tags in chlamydomonas using 3'-RACE.
BACKGROUND
The 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.


RESULTS
Here, 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..


CONCLUSIONS
We propose that our 3'-RACE FST method can be used to build large scale FST libraries in Chlamydomonas and other transformable organisms.
Present address : Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7139/Université Pierre et Marie Curie, Station Biologique de Roscoff, Roscoff, 29280, France. ovallon@ibpc.fr.
Laurence
L
Meslet-Cladière
Olivier
O
Vallon