| The Chlamydomonas FLA14 gene encodes dynein light chain 8 (Q39580), which is a common subunit of several flagellar complexes, including outer arm dynein, inner arm dynein I1/f, the radial spokes, and cytoplasmic dynein 1b (the retrograde motor for intraflagellar transport). LC8 thus is abundant in flagella, and its gene is strongly induced (~5X) by deflagellation. Among genes encoding flagellar proteins, FLA14 is unusual in that it lacks introns. This suggested that FLA14 might serve as the basis for a vector for the expression of cDNAs in Chlamydomonas . To test this, regions upstream and downstream of the FLA14 coding region were cloned to flank a polylinker site and triple HA tag; the ble gene was inserted upstream of the FLA14 cassette as a selectable marker for transformation. cDNAs encoding different flagellar proteins were inserted individually into the vector and then transformed into mutants for the corresponding genes. Transgene expression was observed with a high frequency, and reached levels sufficient to rescue the mutants. After deflagellation, expression of the transgenes was induced 5-10 fold, indicating that the vector mimics the expression pattern typical for many flagellar genes. So far, expression of the transgene has remained stable for at least 2 years. Successful expression and rescue also were obtained when the HA tag was replaced by a GFP tag. Therefore, the FLA14-based vector is a useful tool for mutant rescue and epitope-tagging of flagellar proteins via expression of their cDNAs.
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