Figure 4

Integration sites in A. pretiosum transconjugants of pAP40, pAP42 and pAP50. Total DNA from apramycin resistant transconjugants were digested with ApaI and hybridized to a DIG-labeled IS117 probe. Unintegrated pAP40, pAP42 and pAP50 from E. coli were similarly digested and included as controls. Patterns of hybridized bands of each sample representing an integration site was arbitrarily labeled A, B, C and D. Site A consisted of an unchanged 3.5 kb band from the right arm of the integrated plasmid, and a variable left arm, dependent on the cloned gene of interest. Control plasmids have the same molecular weight as the left arm of its corresponding integrated plasmids because of an ApaI site on the genome, and not because of unintegrated plasmids in the total DNA extract. Transconjugants 1, 2, 3 and 4 of pAP40, pAP42 and pAP50 were subsequently tested for AP3 production.