FIGURE 5.

Precise manipulation of pGKL VLEs in vivo revealed an essential role of pGKL promoters in mRNA capping and non-template-based 5′ polyadenylation. (A) A closer view of the native pGKL2 region subjected to homologous recombination with a PCR cassette depicted in Supplementary Figure S5 shows a tightly packed VLE genome. The 3′ end of the K2ORF3 coding region overlaps the K2ORF2 promoter, 5′ UTR, and the first four nucleotides of the K2ORF2 coding region. The pGKL2 VLEs displayed in (B,C) are in the reverse orientation of those in Figure 1. Shades of gray indicate the degree of transcript capping, as shown in Figure 1. (B) A PCR cassette containing an antibiotic resistance gene (G418) under the control of the ORF2 promoter from pGKL1 (K1UCR2) and the ORF1 promoter from pGKL1 (K1UCR1) (Supplementary Figure S5) was inserted into the K2ORF2 promoter region by homologous recombination in vivo. The resulting VLE, pRKL2-1, contains two genes, aminoglycoside 3′-phosphotransferase (coding for G418 resistance) and K2ORF2, that are artificially controlled by the pGKL1 promoters K1UCR2 and K1UCR1, respectively. Shades of gray indicate the degree of transcript capping, as shown in Figure 1. The 5′ RACE results of pRKL2-1-encoded mRNAs are summarized in the text and in Supplementary Table S7. (C) Electrophoretic analysis of pGKL VLEs in K. lactis clones. M, lambda DNA/Eco130I (StyI) marker (Fermentas); lanes 1 and 4, native pGKL VLEs from K. lactis IFO1267 (pGKL1 [8874 bp] is labeled with an asterisk, and pGKL2 [13447 bp] is labeled with an arrow); lane 2, linear VLEs purified from K. lactis IFO1267 carrying both the recombinant (higher MW) and wild-type pGKL2 VLEs; lane 3, linear VLEs purified from K. lactis IFO1267 containing the recombinant pRKL2-1 VLE (14353 bp). The shorter wild-type pGKL2 was lost after cultivation for ≈60 generations in selective medium containing G418.