THE CHLAMYDOMONAS CHLOROPLAST CLPP GENE CONTAINS TRANSLATED LARGE INSERTION SEQUENCES AND IS ESSENTIAL FOR CELL-GROWTH

Sequence determination of the chloroplast clpP gene from two distantly related Chlamydomonas species (C. reinhardtii and C. eugametos) revea led the presence of translated large insertion sequences (IS1 and IS2) that divide the clpP gene into two or three sequence domains (SDs) an d are not found in homologous genes in other organisms. These insertio n sequences do not resemble RNA introns, and are not spliced out at th e mRNA level. Instead, each insertion sequence forms a continuous open reading frame with its upstream and downstream sequence domains. IS1 specifies a potential polypeptide sequence of 286 and 318 amino acid r esidues in C. reinhardtii and C. eugametos, respectively. IS2 encodes a 456 amino acid polypeptide and is present only in C. eugnmetos. The two Chlamydomonas IS1 sequences show substantial similarity; however, there is no significant sequence similarity either between IS1 and IS2 or between these insertion sequences and any other known protein codi ng sequences. The C. reinhardtii clpP gene was further shown to be ess ential for cell growth, as demonstrated through targeted gene disrupti on by particle gun-mediated chloroplast transformation. Only heteropla smic transformants could be obtained, even under mixotrophic growth co nditions. The heteroplasmic transformants were stable only under selec tion pressure for the disrupted clpP, rapidly segregated into wild-typ e cells when the selection pressure was removed, and grew significantl y more slowly than wildtype cells under phototrophic conditions.