FUNCTIONAL INVIVO ANALYSES OF THE 3' FLANKING SEQUENCES OF THE CHLAMYDOMONAS CHLOROPLAST RBCL AND PSAB GENES

Possible roles of untranslated sequences at the 3' ends of chloroplast genes, which include inverted repeat elements, were investigated in C hlamydomonas reinhardtii in vivo. Chlamydomonas chloroplast rbcL or ps aB 3' flanking regions were coupled in various arrangements 3' to a ch imeric gene consisting of a Chlamydomonas chloroplast atpB promoter se quence fused 5' to the Escherichia coli uidA (GUS) structural gene. Th ese genes were introduced into the Chlamydomonas chloroplast genome at the same location by homologous recombination following microprojecti le bombardment. Transformants harboring chimeric GUS genes fused to rb cL or psaB gene 3' inverted repeat sequences in their normal forward o rientations accumulated GUS transcripts of a single size, whereas GUS transcripts of heterogenous sizes accumulated in transformants harbori ng the same gene lacking an inverted repeat sequence at its 3' end. Th us, the 3' flanking regions of the rbcL and psaB genes can define the location of the 3' terminus of a transcript in vivo. In chloroplast tr ansformants harboring chimeric GUS genes fused to multiple inverted re peat sequences in their normal forward orientations, only GUS transcri pts accumulated that were terminated by the first inverted repeat sequ ence. The latter data suggest that the 3' ends of these RNAs are the p roducts of either transcription termination or endonucleolytic cleavag e. Analyses of GUS transcripts in transformants harboring GUS genes te rminated by rbcL or psaB gene 3' flanking regions in reversed orientat ions indicate that transcript 3' end formation in vivo requires nucleo tide sequences located outside the inverted repeat elements. Inasmuch as decay rates of GUS transcripts were found to be independent of the presence of a 3' inverted repeat sequence, RNA stabilization does not appear to be a major in vivo function of these elements in the Chlamyd omonas chloroplast transcripts studied.