Notes: In this paper, Tfl DNA polymerase was used for two-step RT-PCR. The amplified fragments were cloned into the pGEM®-T Vector. Promega's Terminal Deoxynucleotidyl Transferase (TdT) was used to end label specific oligos for screening a frog brain cDNA library. T7 and T3 RNA polymerases were used to make digoxigenin labeled riboprobes for in situ hybridization. (1281)

Notes: Enzymatically active diphtheria toxin was formed in vitro by transcription/translation from PCR products carrying the A- and B-fragments of the toxin. T3 RNA Polymerase was used for in vitro transcription and the Rabbit Reticulocyte Lysate System was used for translation of proteins. Both wild type and mutant proteins were produced and assayed. The synthesized proteins were tested for trypsin sensitivity, ability to inhibit protein synthesis, binding and translocation in Vero cells. Considerable alterations can be made to the C-terminal region of the protein without blocking translocation. Some mutant toxins were less toxic than wild type toxins in unnicked form, but when nicked most exhibited the same toxicity. (2226)