A Modified Screening System for Loss-of-Function and Dominant Negative Alleles of Essential MCMV Genes
Figure 5
Construction and CCA complementation of pM99 mutants.
(A) Flpe-NIH were nucleofected with the indicated BAC and plasmids and plaque formation quantified at 6 dpt. F, Flag-tag; P, PM99. Depicted are mean and SD of duplicate samples of two independent experiments. (B) The amino acid sequences of 34 pUL11 homologues (accession numbers are listed in Table S2) were aligned using the Vector NTI AlignX program (Invitrogen) via the BLOSUM 62 similarity matrix. The similarity plot was calculated using a 5 amino acid window size, with scores for weak and strong similarity and identity of 0.2, 0.5, and 1.0, respectively. The x axis represents the number of amino acids in the consensus sequence. The conserved region (CR) is indicated below the diagram. (C) Schematic overview of the pM99 mutants. Proteins are depicted as gray bars, the conserved region (CR1) are indicated as black boxes. Deletion mutants are shown with bridged spacing. Numbers on the right indicate the amino acid of pM99 affected by the mutation. (D) Flpe-NIH cells were treated as in (A). Mutants are based on the PM99-igfp plasmid. Depicted are mean and SD of duplicate samples of two independent experiments.