The isolation of cell surface mutants of RAG-1 was demonstrated by a method based on the differential immunoprecipitating properties of wild-type cells and mutants. In the presence of antiserum, about 99% of wild-type cells in suspension sedimented following overnight agglutination. The kinetics of antiserum-induced agglutination was quantified by measuring the time-dependent decrease in turbidity. For mutant isolation, a sample of the cells remaining in the supernatant fluid served as inoculum for a fresh culture. This enrichment procedure of sequential growth and agglutination was repeated until the supernatant fluid following the overnight agglutination step remained turbid. Of the 40 colonies isolated in this manner, 34 exhibited altered agglutination properties.

The agglutinability of wild-type cells varied with the phase of growth - cells harvested during the exponential phase were agglutinated better than those in stationary phase. In contrast, the antiserum-induced agglutination of most mutants was poor during the early phases of growth, but improved during late-stationary phase. One mutant failed to agglutinate in the presence of antiserum irrespective of the phase of growth.


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