Marker rescue mapping of the combined Condit/Dales collection of temperature-sensitive vaccinia virus mutants

Abstract

Complementation analysis of the combined Condit/Dales collection of vaccinia virus temperature-sensitive mutants has been reported (Lackner, C.A., D'Costa, S.M., Buck, C., Condit, R.C., 2003. Complementation analysis of the Dales collection of vaccinia virus temperature-sensitive mutants. Virology 305, 240-259), however not all complementation groups have previously been assigned to single genes on the viral genome. We have used marker rescue to map at least one representative of each complementation group to a unique viral gene. The final combined collection contains 124 temperature-sensitive mutants affecting 38 viral genes, plus five double mutants.

Fig. 1. Coarse marker rescue mapping of Cts13

A) A HindIII map of the vaccinia genome showing the positions of large sized (YE) PCR fragments and intermediate sized (LM) PCR fragments. B) Marker rescue of Cts13. Dishes were infected with Cts13, transfected with pools of PCR fragments as indicated, incubated at 39.7°C for 4 days, and stained with crystal violet.

Fig. 2. Refined marker rescue mapping of Cts13

A) A map of a portion of the vaccinia genome containing the HindIII D-A junction, showing the positions of YE 7, and the LM fragments contained within YE 7. B) Marker rescue of Cts13. Dishes were infected with Cts13, transfected with pools of PCR fragments as indicated, incubated at 39.7°C for 4 days, and stained with crystal violet. LM all contains LM fragments 25–34, covering YE 7 and YE 8.

Fig . 3. Gene specific marker rescue mapping of Cts13

A) A map of a portion of the HindIII A fragment of the vaccinia genome. The positions of genes (arrows) and LM 28 are indicated. B) Marker rescue of Cts13. Dishes were infected with Cts13, transfected with pools of PCR fragments as indicated, incubated at 39.7°C for 4 days, and stained with crystal violet.

Fig. 4. DNA sequence of Dts40

The nucleotide sequence encoding the overlap between the carboxy terminus of J3R and the amino terminus of J4R is shown, with respective translations above the sequence. Wild type sequence is shown at the top and the Dts40 mutant sequence is shown at the bottom. The mutant nucleotide in the G to A transition is underlined in each sequence. The single letter amino acid code is positioned above the first nucleotide in the relevant reading frame for each peptide. Dots (...) indicate that the protein continues upstream (J3R) or downstream (J4R).

Fig. 5. Viral DNA replication in mutant infected cells

Cells were infected with the indicated viruses at MOI = 10, incubated at 31°C (♦) or 39.7°C (■) and assayed for viral DNA by slot blot hybridization with a radiolabeled vaccinia specific DNA probe as described in materials and methods.