A new method to identify flanking sequence tags in chlamydomonas using 3’-RACE
Abstract
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.. CONCLUSION: We propose that our 3'-RACE FST method can be used to build large scale FST libraries in Chlamydomonas and other transformable organisms.
Related
- Method to Identify Liver-to-Brain Region-Specific Crosstalk Using TurboID
- Optimized Epitope-Flanking Sequences Enhance Antigen Presentation and T-Cell Responses Following mRNA Vaccination.
- A simple and accurate method for inferring missing ploidy information from sequence data
- All in this together: Multifactorial stress combination promotes cell aggregation in Chlamydomonas reinhardtii
- The enemy of my enemy: A bacterial antagonist rescues Chlamydomonas by inactivating a toxin from a rival
BibTeX
@article{mesletcladiere2026method,
title = {{A new method to identify flanking sequence tags in chlamydomonas using 3’-RACE}},
author = {Laurence Meslet-Cladière and Olivier Vallon},
journal = {Plant Methods},
year = {2026},
month = oct,
doi = {10.1186/1746-4811-8-21},
url = {https://hal.science/hal-05839481v1},
note = {CC BY},
}
RIS
TY - JOUR
TI - A new method to identify flanking sequence tags in chlamydomonas using 3’-RACE
AU - Laurence Meslet-Cladière
AU - Olivier Vallon
PY - 2026
DA - 2026/10/26
JO - Plant Methods
DO - 10.1186/1746-4811-8-21
UR - https://hal.science/hal-05839481v1
AB - 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.. CONCLUSION: We propose that our 3'-RACE FST method can be used to build large scale FST libraries in Chlamydomonas and other transformable organisms.
SN - 1746-4811
ER -