Protein Synthesis in Chlamydomonas reinhardi: 1. Characterization of Temperature-Sensitive Mutants in Protein Synthesis. 2. Action of the Drug, Chloral Hydrate

Citation

Cross, John William, Jr. (1976) Protein Synthesis in Chlamydomonas reinhardi: 1. Characterization of Temperature-Sensitive Mutants in Protein Synthesis. 2. Action of the Drug, Chloral Hydrate. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/M5NZ-6V45. https://resolver.caltech.edu/CaltechTHESIS:11122009-095851274

Abstract

I. Mutants of Chlamydomonas which are temperature-sensitive in protein synthesis were analyzed. These mutants were previously shown to be unaffected in RNA synthesis or in nucleotide pool levels at the growth-restrictive temperature (33°) although amino acid incorporation is blocked at least 40%. Here it is demonstrated that exposure of these cells to non-permissive conditions causes their polysomes to break down, resulting in the accumulation of 80S ribosomes which are not bound to mRNA. There was no defect detected in either mutant when their rates of charging of any of the 20 amino acids to tRNA were measured at the restrictive temperature in vitro, or in one of them whose degree of charging of tRNA was estimated in vivo at 33°. It is concluded that the defects in these mutants result from defective initiation of protein synthesis in vivo at 33°. II. The mechanism by which the anesthetic, chloral hydrate, inhibits protein synthesis was investigated in Chlamydomonas. This drug was previously shown to inhibit protein synthesis and cell division in Chlamydomonas at concentrations similar to those which produce anesthesia in vertebrates. The incorporation of amino acids into protein is rapidly inhibited after the drug is added to cells. At the same time, polysomes in the cells partially break down into monosomes, which are not bound to mRNA, but do contain nascent peptides capable of reacting with puromycin. Maximum polysome breakdown occurs in 15-30 min and is followed by a gradual reforation of polysomes, which is complete in 4-8 hours. Amino acid incorporation remains maximally inhibited during this period (85-90% inhibition at 10 mM chloral hydrate). The inhibition of protein synthesis by two compounds which are metabolites of chloral hydrate in other organisms was examined. Protein synthesis is inhibited by trichloroethanol, but not by trichloroacetic acid at concentrations of 10 mM. Trichloroethanol produces the same effects on polysomes as does chloral hydrate. However, in Chlamydomonas no significant amount of chloral hydrate is metabolized to either of these two substances, and so it appears that the effects which are observed are due to the action of chloral hydrate itself. A preliminary investigation of the binding of chloral hydrate to cells shows that Chlamvdomonas accumulates the drug to a higher concentration than that found in the medium. III. An effort was made to enable the selection of mutants of Chlamydomonas conditionally defective in assembly of chloroplast membranes. Chlamydomonas cells which had formed active chloroplasts were found to be killed by photooxidation in an intense light beam, and inhibition of photosynthetic electron transport afforded significant protection from this killing. Therefore it was reasoned that Chlamydomonas mutants unable to form photosynthetically active chloroplasts would be protected. It was, however, found that photooxidation does not select for mutants with reducted contents of photosynthetic pigments.

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