Initiation and elongation steps of messenger RNA synthesis are controlled by the activity of RNA polymerase II. The present study tests the hypothesis that tissue hypoxia alters RNA polymerase II activity in the cerebral cortex of fetal guinea pig brain. Twelve pregnant guinea pigs were exposed to 21%(n=6)or 7% (n=6) oxygen for 60 min. Brain cell nuclei were isolated from the cortex of normoxic and hypoxic guinea pig fetuses at term. RNA polymerase activity was determined in a medium containing isolated nuclei, 40 mM Tris-HCl buffer (pH 7.9) 25 mM ammonium sulphate, 2 mM MgCl2, 1.5 mM MnCl2, 0.3 mM ATP, 0.3 mM GTP, 0.3 mM CTP and 1 μCi 3H-UTP. Under these conditions the activity of all three RNA polymerases (bound form) are expressed. RNA polymerase activity was determined in the presence of dA-dT(RNA polymerase free form) and α-amanitin (RNA Polymerase II-amanitin sensitive, and RNA polymerase I + III-amanitin insensitive). In the normoxic brain nuclei, RNA polymerase II and I+III activities were: bound form, 62.9± 5.6, and 141.9 ± 9.4 pmoles/mg protein/hr; free form, 105.64± 8.4, and 45.9 ± 6.4 pmoles/mg protein/hr. In the hypoxic brain nuclei, RNA polymerase II and I+III activities were as follows: (bound form, 95.9 ± 6.9, and 152.4 ± 11.2 pmoles/mg protein/hr; free form, 50.0 ± 5.4, and 10.4 ± 4.2 pmoles/mg protein/hr. The results show that while there is a significant decrease in the RNA polymerase II and I+III (free form) activity in the hypoxic brain nuclei (p<0.005), there is a significant increase in RNA polymerase II activity (bound form) as compared to normoxic group (p<0.005). These data suggest that brain tissue hypoxia alters the activity and distribution of of RNA polymerases. We conclude that increased activity of RNA polymerase II (bound form) may increase the transcriptional activity and is a potential mechanism of hypoxia-induced alteration of gene expression. (Funded by NIH-HD-20337, MOD #6-FY94-0135, UCPR 506-93)
