High Frequency Re-isolation of Intact Integrative Plasmids in Penicillium chrysogenum
Bañuelos, O.; Naranjo, L.; Casqueiro, J.; Gutiérrez, S.; Martín de Valmaseda, E.; Ullán, R.V.; and Martín, J.F.
Area
de Microbiología, Facultad de Biología, Universidad de León,
24071 León,
Instituto de biotecnología (INBIOTEC), Avda. del Real Nº1,
24006 León.
The efficiency of co-transformation in P. chrysogenum Wisconsin 54-1255 pyrG- and the fate of the transforming DNA was studied using an integrative (pEF43) and an autonomous replicating plasmid (pAM9L). The results showed a co-transformation frequency of near 70% of all transformants tested. The total efficiency of transformation in co-transformation experiments was shown to be dependent on the plasmid marker used in the transformant selection (i.e. markers in the integrative or autonomous replicating vector). Analysis of the plasmids re-isolated from several co-transformants showed that different populations of plasmids coexist in the fungal host. Interestingly, in all co-transformants studied, the integrative plasmid was found to be replicating autonomously without integrating into the host genome. In some cases, co-integrates were formed by recombination between autonomous replicating (pAM9L) and integrative (pEF43) plasmids. However, unexpectedly, the non-reorganized pEF43 integrative plasmid used in the co-transformation assays was rescued from some co-transformants, suggesting that it replicates autonomously when it is accompanied in the cell by pAM9L. These results suggest that interaction between the integrative vector and the autonomous-replicating sequence AMA1 of the pAM9L may provide functions for replication of the integrative plasmid.
