Worm Breeder's Gazette 9(3): 87
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
In order to determine the onset of transcription, we have developed a method for measuring RNA synthesis in developing embryos. The Cowan and Mclntosh (Cell 41, 923, 1985) procedure for isolating and permeabilizing embryos was modified by substituting hypochlorite digestion for homogenization of the worms to yield cleaner preparations in which the embryos do not clump together. Approximately 70% of the embryos passed through a shortened Hamilton needle were rendered permeable to the dye Nile blue, and this permeability persisted for at least 2h. Early cleavage patterns, E.a and E.p cell migrations, and gut granule formation were usually normal in permeabilized embryos, although abnormal early cleavages followed by early arrest (less than 100 cells) were observed in a few instances. The culture medium used in these studies was a slightly modified version of that designed by Edgar and McGhee 114: 109, 1986) which supports normal development through hatching in embryos permeabilized by rupturing the vitelline membrane with a micro-manipulator. Mixed-stage populations of permeabilized embryos incubated in [3H]uridine showed a linear rate of label incorporation into trichloroacetic-acid-precipitated RNA of approximately 20 pg/embryo/h. This incorporation is completely inhibited by 10 ug/ml actinomycin D. Autoradiograms of embryos labelled in this manner and fixed with either Carnoy's or glutaraldehyde reveal differential silver grain densities over different embryos, often with nuclei labelled more intensely than cytoplasm. Greater levels of embryo labelling were correlated with later developmental stage. Since grain densities above background were observed with labelling times as short as 10 min, an accurate pinpointing of the stage at which transcription begins should be possible. Unfortunately, we were unable to complete these experiments in the time allotted. The development of these methods will also make possible determination of the functions of early zygotic transcription. With embryos permeabilized in the presence of actinomycin D or -amanitin, observed abnormalities in embryogenesis not attributable to side- effects would be direct or indirect consequences of the absence of zygotic gene expression. The assay for RNA synthesis in mixed-stage embryo cultures presented here allowed us to determine the lowest drug concentration at which maximal transcription inhibition is achieved; side-effects of the drug should be minimized at this concentration. For actinomycin D, 1 ug/ml was found to be the lowest concentration for which 100% inhibition of [3H]uridine incorporation into RNA was attained by the end of the first 30 min interval (total incorporation during this interval was approximately 30% that of uninhibited cultures). If the appropriate concentration of -amanitin were similarly determined, the mutant ama-1, with an RNA polymerase 11 150 times less sensitive to the drug, would provide an elegant control for side-effects not caused by inhibition of mRNA synthesis.