main, but lacks clear BH2, BH3, or BH4 domains (5–7). The HV68 v-bcl-2 also shares with bcl-2 and v-bcl-2 proteins a hydrophobic COOH-terminal domain (5–10). Presuming the functional importance of conserved regions of the protein, we constructed two HV68 mutants with stop codons and frame shift mutations in the v-bcl-2 ORF (v-bcl-2.Stop1, v-bcl-2.Stop2) in which neither the BH1 domain nor the COOH-terminal hydrophobic domain should be translated (Fig. 1). The Stop1 mutation deletes the predicted BH1 domain and inserts a stop codon after 76 amino acids of the predicted 171 amino acid v-bcl-2 (Fig. 1 B). v-bcl-2.Stop2 contains a mutation that inserts a stop codon and frame shift mutation after 10 amino acids of the v-bcl-2 (Fig. 1 B). We generated two independent clones of each construct, indicated as A and B. The genomic structure of v-bcl-2.Stop1A and 1B, and v-bcl-2.Stop2A and 2B were confirmed by Southern blot analyses (Fig. 1 C). Western blot analysis showed that v-bcl-2.Stop viruses express v-cyclin normally in lytically infected 3T12 fibro- blasts (Fig. 1 D). Figure 1. Genomic structure of v-bcl-2.Stop mutant viruses. (A) Schematic representation of the region of the HV68 genome encoding the v-bcl-2 gene (M11 ORF; genomic coordinates 103,418–103,930) (reference 5), the structure of v-bcl-2 mutant viruses and the v-bcl-2 region Southern blot probe (genomic coordinates 101,654–105,377) (reference 5). The shaded region within ORF72 in the v-cyclin.LacZ virus represents a LacZ cassette insertion between 102,704 bp and 103,179 bp (reference 18). The crosshatched region in the M11 ORF in v-cyclin.LacZ signifies the part of the M11 ORF de- leted in v-bcl-2.Stop1. (B) Schematic representation of the v-bcl-2 ORF showing the position of the conserved BH1 domain and the sites of Stop1 and Stop2 mutations in the v-bcl-2 ORF. Shaded regions are the portions of the ORF predicted to be translated. (C) Southern analysis of wt HV68, v-bcl-2.Stop1 and Stop2 mutants, v-cyclin.LacZ, and v-cyclin.MR viruses. Purified genomic viral DNA (1 g) from the indicated viruses was digested with XhoI and BamHI (lanes 1–5) or NheI and BamHI (lanes 6–10) and Southern analysis performed with the v- bcl-2 region probe (A; references 18 and 29). Expected fragment sizes for XhoI and BamHI digests: lane 1/wt HV68, 5.2 kb; lane 2/v-cyclin.LacZ mutant, 3.8 kb and 1.1 kb; lane 3/v-bcl-2.Stop1A mutant, 3.3 and 1.9 kb; lane 4/v-bcl-2.Stop1B mutant, 3.3 and 1.9 kb; lane 5/v-cyclin.MR, 5.2 kb. Expected fragment sizes for NheI and BamHI di- gests: lane 6/wt HV68, 5.2 kb; lane 7/v-cyclin-LacZ mutant, 3.8 kb and 1.5 kb; lane 8/v-bcl-2.Stop2A mutant, 3.5 and 1.7 kb; lane 9/v-bcl-2.Stop2B mutant, 3.5 and 1.7 kb; lane 10/v-cyclin.MR; 5.2 kb. (D) Immunoblot analysis of wt HV68, v-bcl-2.Stop1 and Stop2 mutants, v-cyclin.LacZ, or v-cyclin.MR virus infected NIH 3T12 cell lysates for v-cyclin and -actin protein expression. 933 Gangappa et al.