Figure 1. DNAI1 mutants loose part of their outer dynein arms.
A. The two alleles of the DNAI1 gene were successively replaced via homologous recombination by the blasticidin (BLA) and neomycin (NEO-G418) resistance marker coding sequences, flanked by the DNAI1 flanking sequences (black bars), to produce the DNAI1 mutant strain. The relative positions of the PCR primer pairs used for amplification of the inserts (F1 and R1 in orange) and integration control (F2 and R2 in red) are indicated.
B. PCR with the primers F2 and R2, designed to hybridize outside the first 100 bp of the DNAI1 flanking sequences used for recombination, was performed on 100 ng of genomic DNA from WT, DNAI1 mutant subclones 1 to 6 (M1 to M6) and cells transformed with only one insert or the other (B1 and B2 for blasticidin-resistant subclones, and G1 and G2 for neomycin/G418-resistant subclones). The expected resistance marker cassettes were detected in all the different clones for each construct (596 bp for BLA and 1067 bp for NEO/G418). In addition, a band detected in all mutant types and clones, whatever the order of the transformation process, and with the same size as the full DNAI1 WT gene (2165 bp), was sequenced and proved to correspond to an extracopy of the gene.
C. RT-PCR targeting the DNAI1 coding sequence was performed on total RNA extracts from WT parasites, simple (B2) and double (M1 and M2) DNAI1 mutant subclones, as well as AB cells. In parallel, the same samples were processed for RT-PCR targeting FLA1 mRNA and for direct PCR targeting FLA1 DNA as positive and negative controls respectively. Internal negative controls with H2O for RT-PCR and positive control with genomic DNA for PCR are shown in the last column (+/−).
D. Cell cytoskeletons were extracted by detergent treatment and processed for transmission EM. Flagella cross-sections were analysed for each strain. Asterisks indicate microtubule doublets where outer dynein arms are missing. Scale bar represents 200 nm.
E. Numbers of missing outer dynein arms were quantified in transmission EM pictures of detergent-treated flagellum cross-sections and plotted as percentage of the total observed cross-sections for each strain.
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