Suppression of a +1 T mutation by a nearby substitution in the mitochondrial cox1 gene of Chlamydomonas reinhardtii : a new type of frameshift suppression in an organelle genome

Abstract

In Chlamydomonas reinhardtii, mutants defective in the cytochrome pathway of respiration lack the capacity to grow under heterotrophic conditions (in darkness on acetate). In the dark- strain duM18, a + 1 T addition in a run of four Ts, located at codon 145 of the mitochondrial cox1 gene encoding subunit I of cytochrome c oxidase, is responsible for the mutant phenotype. A leaky revertant (su11) that grows heterotrophically at a lower rate than wild-type cells was isolated from dum18. Its respiration sensitivity to cyanide was low and its cytochrome c oxidase activity was only 4% of that of the wild-type enzyme. Meiotic progeny obtained from crosses between revertant and wild-type cells inherited the phenotype of the mt- parent, showing that the suppressor mutation, like dum18 itself, is located in the mitochondrial genome. In order to map the su11 mutation relative to dum18, a recombinational analysis was performed on the diploid progeny. It demonstrated that su11 was very closely linked to the dum18 mutation less than 20-30 bp away. The cox1 gene of the su11 revertant was then sequenced. In addition to the + 1 T frameshift mutation still present at codon 145, an A-->C substitution was found at codon 146, leading to the replacement of a glutamic acid by an alanine in the polypeptide chain. No other mutations were detected in the cox1 coding sequence. As the new GCG codon (Ala) created at position 146 is very seldom used in the mitochondrial genome of C. reinhardtii, we suggest that the partial frameshift suppression by the nearby substitution is due to an occasional abnormal translocation of the ribosome (+ 1 base shift) facilitated both by the run of Ts and the low level of weak interaction of alanyl-tRNA.

An Abundant Evolutionarily Conserved CSB-PiggyBac Fusion Protein Expressed in Cockayne Syndrome

“…Similarly, the existence and varying efficiency of programmed +1 and −1 frameshifting [64] suggests that frameshift mutations may sometimes be subject to a compensatory ribosomal frameshift that partially preserves the original reading frame. Indeed, ribosomal frameshifting is strongly dependent on context [65] which appears to be very “slippery” in the case of the +T1359 mutation (TTT TTC CCA to TTT TTT CCC) and could in principle increase the frequency of +1 frameshifts. Of course, leaky terminators and weak frameshifts might have been expected to rescue expression of both the CSB-PGBD3 fusion and full length CSB protein, but it should be kept in mind that the CSB-PGBD3 and CSB mRNAs are alternatively spliced and polyadenylated transcripts with different intron/exon structures and different 3′ UTRs.…”

Mitochondrial Genetic Manipulation

“…The mutants lack the cytochrome pathway of the mitochondrial electron transport chain, but their respiratory activity is partially maintained via the alternative oxidase. Phenotypically, the mutants have lost their capacity to grow under heterotrophic conditions (darkness and addition of acetate as a reduced carbon source), whereas their photoautotrophic growth is barely affected (Remacle et al 1998). These characteristics allow selection of mitochondrial transformants by restoration of the capacity to grow in the darkness.…”