KINETIC-ANALYSIS OF A SIGNAL-TRANSDUCTION PATHWAY BY TIME-RESOLVED SOMATIC COMPLEMENTATION OF MUTANTS

Sensory control of sporulation in Physarum polycephalum plasmodia is m ediated by a branched signal-transduction pathway that integrates blue light, far-red light, heat shock and the starvation state. Mutants de fective in the pathway were isolated and three phenotypes obtained: bl ue-blind? general-blind and light-independent sporulating, When plasmo dia of the blue-blind mutant Blu1 were exposed to a pulse of blue ligh t and subsequently fused to non-induced wild-type plasmodia? the resul ting heterokaryons sporulated, indicating a functional blue-light phot oreceptor in the mutant. When the general-blind mutant Nos1 was fused to a wild-type plasmodium which had been induced by light, sporulation of the heterokaryon was blocked. However, the dominant inhibition of sporulation by Nos1 was gradually lost with increasing time between in duction by light and time of fusion, suggesting that Nos1 can be bypas sed by the time-dependent formation of a downstream signal-transductio n intermediate. Phenotype expression in constitutively sporulating (Co s) mutants depended on starvation, The Cos2 product was titrated by fu sing mutant plasmodia of different sizes to wild-type plasmodia of con stant size and analysing the sporulation probability of the resulting heterokaryon. The titration curve indicates that a small change in the amount of Cos2 product can cause sporulation. We conclude that somati c complementation analysis allows the time-resolved evaluation of the regulatory function of mutations in a signal-transduction pathway with out prior cloning of the gene. This shortcut allows us to characterize many mutants quickly and to select those for molecular analysis that display a well-defined regulatory function.