Correct splicing of a group II intron from a chimeric reporter gene transcript in tobacco plastids

Abstract

A in vivo test system was developed to study group II Intron splicing in higher plant chloroplasts. The chimeric reporter gene uldA* was constructed by translatlonal fusion of an intron-containlng segment of the plastld atpF gene with the coding region of a plastld uldA reporter gene. The chimeric uldA* gene was inserted into the tobacco plastid genome by the biollstlc transformation procedure using a plastid targeting vector. Correct intron excision was confirmed by Northern blot analysis, by sequencing amplified DNAs and by accumulation of the encoded fi-glucuronldase (GUS), the expression of which was dependent on intron removal. Removal of the intron from the uldA*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 highlyexpressed uldA* mRNA is not due to depletion of factors) required for the atpF intron removal. A derivative of uldA*, with a stop codon introduced into the loop of domain VI, was also tested. The mutations did not affect the splicing efficiency. The chimeric uldA* splicing system will facilitate the study of structural and sequence requirements for group II Intron splicing in plastids of higher plants. © 1995 Oxford University Press.