Related Experiment Videos
Mutation of a cytoplasmic gene in Chlamydomonas alters chlorplast ribosome function
Abstract:
A mutation, car, determining resistance to several macrolide antibiotics, including carbomycin, has been identified in the alga Chlamydomonas as cytoplasmic, and mapped in the known cytoplasmic linkage group close to genes determining resistance to other antibiotics, including streptomycin, erythromycin, and spectinomycin. The effect of the car mutation on chloroplast ribosome function was demonstrated with an in vitro system incorporating amino acids especially developed to assess activity of 70S chloroplast ribosomes. In an S-30 extract containing both 70S chloroplast and 80S cytoplasmic ribosomes, low concentrations of Mg(++) and spermidine favored 80S ribosome activity, and high concentrations activated 70S ribosomes and reversibly inactivated the 80S component. Under conditions favoring chloroplast ribosome activity, carbomycin inhibited incorporation by an S-30 extract, and by purified 70S ribosomes from wild-type but not from car cells. These results show that cytoplasmic genes are directly involved in chloroplast ribosome function and they suggest that the car gene product is a ribosomal protein; the results further strengthen the evidence that the cytoplasmic linkage group is located in chloroplast DNA.
Insights
A Chlamydomonas mutation (car) confers macrolide antibiotic resistance by affecting chloroplast ribosome function. This finding demonstrates cytoplasmic gene involvement in chloroplast ribosomes, suggesting the car gene encodes a ribosomal protein.
Area of Science:
- * Molecular Biology
- * Genetics
- * Biochemistry
Background:
- * Antibiotic resistance is a growing concern in microbial systems.
- * Chloroplasts possess their own protein synthesis machinery (70S ribosomes) distinct from cytoplasmic ribosomes (80S).
- * Understanding the genetic basis of chloroplast ribosome function is crucial for various biological processes.
Purpose of the Study:
- * To investigate the effect of a specific mutation (car) on chloroplast ribosome function in Chlamydomonas.
- * To determine if cytoplasmic genes are involved in chloroplast ribosome activity.
- * To elucidate the nature of the car gene product and its role in antibiotic resistance.
Main Methods:
- * Genetic mapping of the car mutation within the Chlamydomonas cytoplasmic linkage group.
- * In vitro biochemical assays using S-30 extracts and purified 70S chloroplast ribosomes.
- * Assessment of amino acid incorporation under varying Mg(++) and spermidine concentrations.
- * Testing the effect of carbomycin on ribosome activity in wild-type and car mutant cells.
Main Results:
- * The car mutation, conferring resistance to carbomycin and other macrolides, was mapped to the cytoplasmic linkage group.
- * Optimized conditions (high Mg(++) and spermidine) selectively activated 70S chloroplast ribosomes.
- * Carbomycin inhibited protein synthesis in wild-type chloroplast ribosomes but not in car mutant ribosomes.
- * The car mutation directly impacts chloroplast 70S ribosome function.
Conclusions:
- * Cytoplasmic genes play a direct role in the function of chloroplast ribosomes.
- * The car gene product is likely a ribosomal protein component of the 70S chloroplast ribosome.
- * These findings support the localization of the cytoplasmic linkage group within chloroplast DNA.