New tools for insertional mutagenesis
Laurence Meslet-Cladière, and Olivier Vallon
Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France
In order to create a new antibiotics resistance marker for Chlamydomonas nuclear transformation, we have recoded, based on codon usage in the nuclear genome, the AadA marker that has been used so extensively in chloroplast transformation. When placed under control of the HSP70A-RBCS2 hybrid promoter and preceeded by the RbcS2 chloroplast targeting peptide, CrAadA confers resistance to spectinomycine and streptomycine. Resistance is stable for several month in the absence of selection pressure. In order to obtain a CrEcAadA shuttle marker that would be usable both in E. coli and Chlamydomonas, we have placed an artificial bacterial promoter and Shine-Dalgarno sequence in frame within CrAadA protein. The resulting CrEcAadA marker can be useful for example in complementation studies in Chlamydomonas, when it is necessary to select transformants before screening. We have introduced CrEcAadA and the classical aphVIII marker into BAC clones, using a non-replicating suicide vector where the marker is flanked by DNA present in the BAC. Both the pop-in and pop-out steps of this procedure are made easier by the use of such a shuttle marker. To facilitate functional genomics studies, we are also seeking to develop easier ways to identify Flanking Sequence Tags (FSTs) in insertion libraries. We have noticed that the CrAadA and aphVIII markers yield almost as many transformants when their promoter or 3'-UTR regions are removed. In the latter case, the signal for cleavage and polyadenylation of the mRNA must be provided by the flanking sequence which therefore becomes part of the 3'-UTR. We have used 3'-RACE to sequence the 3'-UTRs of such transformants. We are fine-tuning the transformation conditions to minimize the occurrence of religation events that we find can prevent retrieval of a true Chlamydomonas FST.
Supported by "Algomics" grant from the Agence Nationale de la Recherche
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