Heterologous genes introduced into the nuclear genome of Chlamydomonas reinhardtii are often poorly expressed. To understand the molecular mechanisms underlying this effect, we examined the influence of various factors on the expression of a chimeric transgene that confers resistance to zeomycin. This marker comprises the bacterial ble gene flanked by 5' and 3' sequences from the Chlamydomonas RBCS2 gene. We found that the frequency with which transformants are recovered is significantly increased when ble is fused to shorter versions of the RBCS2 promoter and when Chlamydomonas introns are introduced into the coding region of ble. The latter effect is particularly evident in the case of the first intron of RBCS2, which dramatically stimulates the transformation frequency and the level of ble expression. We found that this improvement is mediated in part by an enhancer element within the intron sequence, and that this element acts in an orientation-independent manner and is effective when placed either upstream or downstream of the promoter. Our results demonstrate that stable high-level expression of a foreign gene in Chlamydomonas is possible, and highlight a potential role of introns as modulators of gene expression in this alga.
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