Gene targeting to knock out the activity of specific genes has become important due to recent progress in genomics research. But this technique is still unavailable for many organisms, including economically important microorganisms, due to the high background of ectopic integration during genetic transformation. Aspergillus nidulans uvsC is an ortholog of budding yeast RAD51 that plays central roles in recombination-based repair and meiotic recombination. Occurrence of gene targeting at argB locus was under a detection limit in a null mutant of uvsC (less than 5% of control strains) when randomly chosen transformants were examined for their integration profiles. Other differences were also observed in the integration profiles during genetic transformation that were consistent with the expected functions of uvsC. Transcription of uvsC was elevated using the 5Âf sequences of the glyceraldehyde-3-phosphate dehydrogenase (Pgpd) and Taka-amylase A (Ptaa) genes from A. nidulans and A. oryzea, respectively. Maximum of 5-fold increase in the efficiency of targeting was observed at argB, yA, and wA loci when uvsC was under control of Pgpd. Higher level of transcription was achieved with Ptaa, but at the inducible condition with maltose, mycelial growth was significantly suppressed. These results demonstrate that uvsC is involved in integration of extra-cellular DNA integration into chromosomes and is a possible rate-limiting factor for gene targeting. However the increased efficiency of gene targeting is hindered by a deleterious effect of increased transcription on cell proliferation.
Full conference title:
23rd Fungal Genetics Conference
- Fungal Genetics Conference 23rd (2002)