Evidence for an RNA Polymerization Activity in Axolotl and Xenopus Egg Extracts
Figure 1
Axolotl low speed extracts (LSE) polymerize DNA when single-stranded M13 DNA is added to the extracts.
A : Cpm incorporated into TCA precipitable material were registered following 2 hr incubation reactions (44 µL) performed at 20°C in the presence (+) or not (−) of the mentionned DNA template, [α–32P] dCTP as DNA precursor, as well as CaCl2 addition (+) or not (−). LSE prepared from Xenopus UFE and incubated with demembranated sperm nuclei provided the control for dCMP incorporation conditions as well as for activation of LSE by CaCl2 addition. Each histogram corresponded to 2 (Xenopus) to 5 (axolotl) experiments. B : Single-stranded (SS) M13 DNA (150 ng) were mixed in a reaction (88 µL) with axolotl LSE. The reaction was divided into equal aliquots incubated at 20°C during 2 hr (2h, lanes 6) or not (t = 0, lanes 5). A reaction (44 µL, t = 0) without any M13 DNA addition to the extract was also processed (lanes 4). Following phenol/chloroform extractions and ethanol precipitation, the DNA content of each aliquot was analyzed by gel electrophoresis in an 1% agarose gel. Circular M13 DNA either single-stranded (SS; lanes 1 : 100 ng) or double-stranded (DS; lanes 2 : 300 ng) or linearized EcoRI-digested DS M13 (lanes 3 : 400 ng) were electrophorezed in parallel as well as 1 kbp ladder molecular marker (Fermentas, M). Left side : photograph under UV illumination after ethidium bromide staining of the gel. Right side : autoradiograph following gel transfer and hybridization of the corresponding membrane to a radiolabelled SS M13 DNA probe. The migration lengths corresponding to the different forms of M13 DNA are indicated with black diamonds (SS M13), asterisks (supercoiled form of DS M13, I), black squares (closed or nicked relaxed forms of DS M13, II) and black arrows (linearized DS M13, III).