In vivo rearrangement of foreign DNA by Fusarium oxysporum produces linear self-replicating plasmids

Abstract

Particular combinations of fungal strains and transformation vectors allow for fungal rearrangement of normally integrative plasmids, resulting in the creation of linear self-replicating plasmids in Fusarium oxysporum. The rearrangement results in the addition of fungal DNA, including telomere consensus sequences, to plasmid termini. The mechanism by which this rearrangement occurs is unclear, but it has similarities to extrachromosomal gene amplification. A DNA fragment which allows for linear autonomous replication upon reintroduction to the fungus was subcloned and sequenced. This DNA sequence contains the repeated telomeric sequence TTAGGG flanked by a region of twofold symmetry consisting primarily of pUC12 DNA. Isolation and identification of this sequence is the first step toward development of vectors that function as artificial chromosomes in filamentous fungi. This sequence was shown to promote autonomous replication and enhance transformation in several strains of F. oxysporum, Nectria haematococca, and Cryphonectria parasitica.

Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA

“…Chromosomal karyotyping of the uraS auxotrophs used in these experiments showed notable genomic instability and may be a unique characteristic of this strain. Genomic instability due to eventual excision of introduced DNA has been proposed as the apparent mechanism in other fungi (24,29,33). From these previous experiments, it could be inferred that C. neoformans lacks a gene replacement mechanism yet is receptive to ectopic integration which eventually produces rearrangements and/or addition of telomeric sequences with a telomerase, as seen in Paramecium tetraurelia (11).…”

Strategies for the transformation of filamentous fungi

“…Unrelated fungi such as the basidiomycetous yeast Crytococcus neoformans, the dimorphic ascomycete Histoplasma capsulatum, the ®lamentous deuteromycete Fusarium oxysporum (Powell and Kistler 1990) and the taxol-producing ®lamentous fungus Pestalotiopsis microspora (Long et al 1998), can add telomeric repeats to transforming DNA for reasons unknown. Linear transformation vectors containing telomere consensus sequences were created in Fusarium oxysporum by fungal rearrangement of an integrating vector, and functioned with high ef®ciency as autonomously replicating vector in the plant pathogens Nectria haematococca and Cryphonectria parasitica, as well as in F. oxysporum (Powell and Kistler 1990). In Aspergillus nidulans, plasmids containing human telomeric DNA have shown autonomous replication (Aleksenko and Ivanova 1998).…”

A single gene encodes a selective toxin causal to the development of tan spot of wheat.

“…The pCT4 probe hybridized with a single band of -9 kb when genomic DNA was digested with Sacl. The DNA was isolated from the gel, and the 9-kb inserts were used to construct a subgenomic library in the vector pHA1.3 (Powell and Kistler, 1990). Epicurian E. coli XL1 -Blue supercompetent cells (Stratagene) were transformed with the library, and the library was screened by colony hybridization, according to established protocols (Sambrook et al, 1989), with the 3zP-labeled ToxA cDNA insert of plasmid pCT4.…”