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.

Determining the Onset and Early Functions of Zygotic Transcription

M. Costa and K. Kemphues

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 
114: 109, 1986) which supports normal development through hatching 
in embryos permeabilized by rupturing the vitelline membrane with a 
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.