An alternative genetic method to test essential vaccinia virus early genes.
The vaccinia virus F11L gene product was identified during search for additional factors involved in the control of post-replicative viral gene transcription elongation. F11L is a 1065 base pairs (354 aminoacids) gene expressed early during infection with no attributed function. The F11L gene is conserved in many but not all poxviruses. The essential presence of the F11L gene was tested using two different genetic methods. F11L gene disruption by insertion of a selectable cassette containing the Escherichia coli guanine phosphoribosyl transferase gene driven by the viral early-late 7.5K transcriptional promoter resulted exclusively in recombinant viruses containing both the wild type and disrupted alleles, indicating that the F11L gene was essential. However, an alternative test, using transient dominant selection to insert nonsense mutations into the F11L gene, proved that the F11L gene was non-essential for growth in culture. These experiments suggest that misleading results can be obtained using gene insertional mutagenesis as a test of essential presence of the gene. The experiments also provide genetic data on the probability of co-insertion of linked mutations in vaccinia virus genome using transient dominant selection.
Kato
Sayuri E M
SE
Laboratório de Biologia Molecular de Vírus, Instituto de Biofísica Carlos Chagas Filho, CCS, UFRJ, Rio de Janeiro, RJ 21941-590, Brazil.
Greco
Frederico A B
FA
Damaso
Clarissa R A
CR
Condit
Richard C
RC
Moussatché
Nissin
N
D015153:Blotting, Western; D003470:Culture Media; D004926:Escherichia coli; D020043:Genes, Essential; D005821:Genetic Techniques; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D017874:Immediate-Early Proteins / Q000235:genetics* / Q000378:metabolism; D016254:Mutagenesis, Insertional; D011995:Recombination, Genetic; D012641:Selection, Genetic; D014616:Vaccinia virus / Q000235:genetics / Q000254:growth & development*
D003470:Culture Media; D017874:Immediate-Early Proteins; D007041:Hypoxanthine Phosphoribosyltransferase