isatin-beta-thiosemicarbazone and Chromosome-Deletion

We have sequenced and analyzed the transcription of a gene capable of rendering vaccinia virus (VV) dependent upon isatin-beta-thiosemicarbazone (IBT) for growth. Marker rescue analysis of an IBT-dependent mutant of VV, IBTd-1, and a temperature-sensitive mutant of VV, ts56, both of which require IBT to grow at 40 degrees, showed that both lesions mapped to gene G2R. VV mutants with G2R deletions were constructed and shown to also be dependent upon IBT for growth. The nucleotide sequence changes responsible for IBTd-1, ts56, and the gene G2R deletion mutants were determined, and taken together show that IBT dependence results from inactivation of the orf G2R gene product. Gene G2R, which has the capacity to encode a 26-kDa protein, is transcribed solely early during infection. The 1.3-kb mRNA contains a 5' untranslated region of almost 600 nucleotides, and terminates about 20 nucleotides downstream from an early transcription termination signal. Transcription analyses of three flanking genes, as well as the map positions of the VV mutants ts11 and ts60 are also presented.

Topics: Adenine Nucleotides; Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Cell Line; Chromosome Deletion; Chromosome Mapping; Cloning, Molecular; Genes, Viral; Genetic Complementation Test; Isatin; Molecular Sequence Data; Mutation; Oligoribonucleotides; Restriction Mapping; RNA, Messenger; RNA, Viral; Transcription, Genetic; Vaccinia virus; Viral Proteins; Viral Structural Proteins; Virus Replication