Fig. 1.
Scheme of the strategy used to separate TGEV overlapping genes. (A) Structure of the 5′ end of TGEV genes after gene separation by duplication of sequences partially overlapping with the transcription-regulating sequences and introduction of unique restriction sites. Unique endonuclease restriction sites are indicated in italics. Outlined sequences indicate the punctual mutations introduced to generate unique restriction sites. The core sequence (CS) is underlined. The ATG start codon is shown in bold. Duplicated sequences are indicated by dark boxes. TGEV genes are indicated by light boxes. 1b, replicase 1b gene; S, spike gene; E, envelope gene; M, membrane gene; N, nucleoprotein gene. *, Gene Rep 1b termination codon (TGA) and the initiation codon of gene S (ATG) partially overlap. The sequence of the 24 and 83 nt located 5′ upstream of genes S and 3a, respectively, are described in the full-length TGEV genome sequence previously reported (Penzes et al., 2001). (B) Schematic illustration of the full-length TGEV cDNA without (top bar) or with one (pBAC-TGEV-P, pBAC-TGEV-M, pBAC-TGEV-F, and pBAC-TGEV-A), two (pBAC-TGEV-F-Pm, pBAC-TGEV-Pm-A, and pBAC-TGEV-P-M), three (pBAC-TGEV-F-Pm-A), or five (pBAC-TGEV-RS) restriction endonuclease sites. CMV, cytomegalovirus immediate-early promoter; Rep, replicase; An, poly(A) tail of 24 A residues; HDV, hepatitis delta virus ribozyme; BGH, bovine growth hormone termination and polyadenylation sequences.