The isolation and characterization of intact polyribosomes from a cell wall mutant of Chlamydomonas reinhardi.

A technique is described for obtaining intact polyribosomes from a cell-wall mutant of Chlamydomonas reinhardi. When cells were lysed by nonionic detergent in buffers containing high salt and Mg2+-EDTA, polyribosomes of up to 25 ribosomes per polyribosome were obtained on sucrose density gradients. Under these conditions, nascent polypeptide radioactivity was associated with the polyribosomes and not with monoribosomes, and inactive monoribosomes were dissociated to ribosomal subunits. Whole cell lysates contain a mixture of cytoplasmic and chloroplastic polyribosomes. The relative contribution of the two types of polyribosomes was evaluated by polyacrylamide gel electrophoretic analysis of ribosomal RNA extracted from polyribosomes. This analysis showed that less than 15% of the polyribosomes from detergent-lysed cells were from chloroplasts. The contribution of chloroplast polyribosomes to total polyribosomes was increased to about 30% by incubation of the cells in chloramphenicol. When cells were disrupted mechanically (in a French pressure cell) only about 10% of the resulting free polyribosomes were chloroplastic.