Determination of the MDCK RNA pol I promoter transcription initiation site by primer extension analysis. (A) Primer extension reactions on MDCK whole cell RNA using the 32P labeled primers PrimEx1 and PrimEx2. Primer extension products and 32P ΦX174 size marker DNAs (lane M) were subjected to electrophoresis on a 6% polyacrylamide, 7 M urea gel followed by detection of the radioisotope in the gel with a BioRad Molecular Imager Fx. The maximum length of the observed products were approximately 370 bases and 220 bases, respectively, for the reactions using PrimEx1 (lane 1) and PrimEx2 (lane 2). (B) The products from the PrimEx2 reaction were subjected to electrophoresis adjacent to a M13 sequencing ladder on a 6% polyacrylamide, 7 M urea sequencing gel in order to more accurately determine the maximum length of the products synthesized. (C) The MDCK DNA sequences adjacent to the positions where the largest PrimEx2 products terminated.