Identification of insertion sites and deletion boundaries for motility and transcriptional mutants generated with pHyg3
Jason M. Brown, Deborah Cochran, Karl F. Lechtreck and George B. Witman
Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA
Insertional mutagenesis of Chlamydomonas can be used for both forward and reverse genetics. Reverse genetic approaches, which allow one to determine the function of a gene of interest, would be greatly expedited by the availability of a collection of mutants with defined insertions or deletions disrupting all genes of potential interest. Forward genetics, which allows identification of novel genes without prior knowledge of gene function, is expedited if the mutated gene is tagged. For both approaches, identification of the genomic sequences at both ends of inserts would allow the determination of insert locations and the boundaries of any associated deletions. We conducted a pilot mutagenesis using a 1.7 kb fragment of pHyg3 (Berthold et. al., 2002, Protist) and screened for mutants with defects in motility or the regulation of genes encoding flagellar proteins (see accompanying abstract). To facilitate identification of insert-flanking sequences, we optimized the RESDA PCR protocol (González-Ballester et. al. 2005 Anal Biochem) for high-throughput use with pHyg3-generated mutants. To date, 14 motility mutants and 10 putative transcriptional mutants have been analyzed with the optimized protocol. In one round of Hyg RESDA, 13 mutants yielded single PCR products from both ends of the insert and 10 yielded fragments from one end. Sequencing of these products led to the identification of both ends of the insert in 7 strains (30%) and the identification of one end of the insert in 9 strains (39%). Deletion sizes ranged from 4 bp up to 17 kb with most deletions <20 bp. At least five of the motility mutants have insertions or deletions in or near (<5 kb from) genes encoding proteins found in the Chlamydomonas flagellar proteome, three of which are previously uncharacterized. One of these, fap46-1, has a severe ODA-like swimming phenotype that cosegregates with hygromycin resistance in backcrosses.
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