Activity of nuclear DNA-dependent RNA polymerases in mouse limb buds differentiating in vivo or in organ culture.

1. A method was developed to measure the activity of DNA dependent RNA polymerase reactions I (alpha-amanitin-insensitive) and II (alpha-amanitin-sensitive) in homogenates of very small amounts of tissue. This technique was used to study the activity of these polymerase reactions in limb buds from mouse embryos developing in vivo or in an organ culture system. This culture system allows the morphogenetic differentiation of limb buds in early stages of embryonic development (day 10-11 of gestation greater than or equal to 37 - 45 pairs of somites) from a blastema stage to well recognizable cartilaginous bone analgen. 2) An increase in the activities of both types of RNA polymerases was found to occur in vivo in limb buds on day 12 of gestation when compared with the activity measured on day 11 of gestation. A similar increase with a maximum about 24-48h after initiation of the cultures could be observed in the explants differentiating in organ culture. To our knowledge this is the first time that induction processes proceeding in mammalian embryonic tissues could be correlated with an increased activity of RNA polymerases. 3)A rough estimate shows that the rate of transcription due to RNA polymerase I measured in homogenates or in isolated nuclei (measured with substrate concentrations giving about 80% of the maximal rate) would account for the assumed rate of rRNA synthesis in vivo. The measurement of the RNA polymerase activity in isolated nuclei, thus, may give some information about the transcriptional processes occurring under "physiological" conditions. 4) The mammalian organ culture system presented provides a suitable model for studying embryonic differentiation processes.