Journal: Nucleic Acids Research

Article Title: Generalized substitution of isoencoding codons shortens the duration of papillomavirus L1 protein expression in transiently gene-transfected keratinocytes due to cell differentiation

doi: 10.1093/nar/gkm496

Figure Lengend Snippet: Translation of PV Nat L1 genes is preferentially enhanced by aa-tRNA from D8 cultured mouse KCs in a rabbit reticulocyte lysate cell-free in vitro translation system. (A) Both Nat and Mod HPV6b and BPV1 L1 genes from were translated in a cell-free system using rabbit reticulocyte lysate (RRL) in the presence of [ 35 S]methionine over a time course from 15 to 45 min. Production over time of L1 protein from Nat L1 genes (HPV6b or BPV1) is much slower than from Mod L1 genes. (B) Supplementation of the in vitro translation reaction with aa-tRNA from D0 or D8 cultured KC did not affect L1 production from Mod PV L1 genes at 15 min. (C) Supplementation of the in vitro translation reaction with aa-tRNA from D0 or D8 cultured KC enhances L1 production from native L1 genes in a dose-dependent manner from 10 −8 to 10 −6 M at 15 min: inhibition of translation is evident at the highest concentrations of aa-tRNA. Upper panel: representative western blots of three independent experiments. Lower panel: histograms showing means (± S.E.M.) of L1 protein production from six individual blotting assays from three independent experiments quantified by use of a PhosphorImager. Statistical analysis of the results from individual tRNA concentrations from these experiments was conducted using paired t test. P < 0.01 was compared between D0 and D8 aa-tRNA treatments as shown.

Article Snippet: One microgram of each plasmid DNA was incubated with 10 μCi of [ 35 S]methionine (Amersham, Australia) and 20 μl of T7 DNA-polymerase-coupled rabbit reticulocyte lysates (RRL) (Promega, Australia), with or without additional aa-tRNAs as indicated.

Techniques: Cell Culture, In Vitro, Inhibition, Western Blot