CORRECT SPLICING OF A GROUP-II INTRON FROM A CHIMERIC REPORTER GENE TRANSCRIPT IN TOBACCO PLASTIDS

An in vivo test system was developed to study group II intron splicing in higher plant chloroplasts. The chimeric reporter gene uidA* was constructed by translational fusion of an intron-containing segment of the plastid atpF gene with the coding region of a plastid uidA reporter gene. The chimeric uidA* gene was inserted into the tobacco plastid genome by the biolistic transformation procedure using a plastid targeting vector. Correct intron excision was confirmed by Northern blot analysis, by sequencing amplified cDNAs and by accumulation of the encoded beta-glucuronidase (GUS), the expression of which was dependent on intron removal. Removal of the intron from the uidA* mRNA is less efficient (<50%) than from the atpF mRNA (>90%). The efficiency of atpF mRNA splicing is not affected in the plastid transformants indicating that inefficient splicing of the highly-expressed uidA* mRNA is not due to depletion of factor(s) required for the atpF intron removal. A derivative of uidA*, with a stop codon introduced into the loop of domain VI, was also tested. The mutations did not affect the splicing efficiency. The chimeric uidA* splicing system will facilitate the study of structural and sequence requirements for group II intron splicing in plastids of higher plants.