1. Zoospores germinate rapidly and semi-synchronously upon exposure to growth medium or an inorganic salts solution. Amino acid incorporation into protein is detected only after a characteristic lag period, the extent of which is a function of developmental, rather than absolute, time.
2. The ‘turn-on’ of amino acid incorporation occurs after several of the morphological events of germination have taken place - notably, retraction of the flagellum, conversion to a spheroid cell morphology, vesicle fusion with the plasma membrane, formation of the initial cell wall, and elongation of the single mitochondrion. A second group of morphological changes - release of ribosomes from the nuclear cap into the cytoplasm, appearance of multiple mitochondrial profiles, disappearance of the flagellar axoneme, and disappearance of gamma particles - takes place in the cell population during the turn-on of incorporation.
3. Cycloheximide reversibly inhibits germination at a characteristic block point. Inhibited cells accomplish all the known morphological events of germination except the disappearance of the flagellar axoneme and the formation of the germ tube. Amino acid incorporation is inhibited to undetectable levels in growth medium and by about 80% in the inorganic salts medium. Nevertheless, the population kinetics for the morphological changes which occur in cycloheximide-inhibited cells are indistinguishable from those in untreated cells. Cycloheximide effectively enters cells prior to the characteristic block point, since the drug drastically inhibits the low level of amino acid incorporation by zoospores.
4. ‘Pools’ extractable by trichloroacetic acid (TCA) have been examined; the results do not support the possibility of extensive early protein synthesis from endogenous sources.
5. Actinomycin D, at levels which inhibit [3H]uridine incorporation into TCA-precipitable material (nucleic acid) by over 98%, does not affect the population kinetics of germination. The drug does not affect the lag period for amino acid incorporation. The rate of amino acid incorporation (pulse labelling) fluctuates predictably during germination. Actinomycin D only partially inhibits the first ‘round’ of incorporation, but severely inhibits the second.
From this information, we conclude that: (a) the proteins necessary for most of the structural events of germination are preformed in the zoospore; (b) therefore, these events are controlled directly by mechanisms other than differential protein synthesis; (c) two of the known events - disappearance of the flagellar axoneme and formation of the germ tube - do appear to have a requirement for concomitant protein synthesis; (d) germination does not require de novo RNA synthesis; and (e) certain of the first proteins made during germination are evidently synthesized with preformed messages.
Analysis of the survival of mature human eosinophils: interleukin-5 prevents apoptosis in mature human eosinophils
