Replication and expression of a bacterial--mitochondrial hybrid plasmid in the fungus Podospora anserina.

Abstract

Hybrid plasmids consisting of the bacterial plasmid pBR322 and plasmid-like DNA (pl DNA) sequences from the fungus Podospora anserina are not only able to replicate in Escherichia coli but also in the fungus. This was proved by both biophysical and biological evidence involving buoyant density profiles, DNADNA hybridization, and restriction analysis-all confirming that pl DNA behaves as a true plasmid. During its amplification in P. anserina, the hybrid plasmid does not lose its prokaryotic coding capacity as shown after retransfer and subsequent cloning in E. coli P. ansermna is able to express both the eukaryotic and the prokaryotic genetic information of the hybrid plasmid because the occurrence of senescence and the production of .-lactamase could be shown in experiments involving specific hybrid plasmids. In the same systems, it was possible to demonstrate that a hybrid plasmid containing, instead of p1 DNA, a pl DNA homologous region of native mtDNA also could function as a true plasmid. This hybrid plasmid contained about 25% of the genetic information of pl DNA, which corresponds to about 6% of the genetic information of mtDNA. Thus, the data show that hybrid plasmids may be used to shuttle genetic information between P. anserina and E. coli Hence, through the use of a mtDNA replicon, as evidenced by the pl DNA of P. anserina, another pathway in genetic engineering is established.The senescence that occurs regularly in wild strains of the ascomycete Podospora anserina (1) is caused by an infective agent (2) identical with plasmid-like DNA (pl DNA) (3,4). This DNA species is not found in juvenile mycelia and has the following properties: contour length, 0.75 tkm; buoyant density, 1.699 g/ cm3; and molecular size, 2.4 kilobases (kb). Confirmative observations were recently reported by Belcour et al. (5).The pl DNA inserted into the prokaryotic plasmid pBR322 (6) has been cloned in Escherichia coli, and the hybrid plasmid pSP17 that was isolated from the bacterium was able to induce senescence in juvenile cultures of P. anserina (4). Furthermore, heteroduplex analysis and DNA-DNA hybridization (Southern blotting method) have established that pl DNA is an integral part of mtDNA from juvenile mycelia of P. anserina (7,8).The discovery ofpl DNA of mitochondrial origin in a eukaryotic organism has raised the following questions. (i) Is the pl DNA of P. anserina a true plasmid, having the characteristics attributed to bacterial plasmids-i.e., self-replication and expression? The latter has already been shown for pl DNA by induction of senescence in juvenile cultures through transformation experiments (4). Experimental evidence for pl DNA selfreplication will be given in data confirming that pl DNA contains an origin of replication (replicon). Plasmids. Single (pSP4, pSP24) or double (pSP17) copies of pl DNA were integrated in the Sal

Impact of a Disruption of a Pathway Delivering Copper to Mitochondria onPodospora anserinaMetabolism and Life Span

“…Plasmid pSP17 containing two copies of the first intron (pl-intron) of the mitochondrial gene PaCoxI was used for the isolation of the plDNA probe (63 , and incubated at 35°C for 2 h with 20 mg of filter-sterilized Glucanex (Novo Nordisk Ferment AG)/ml. Subsequently, the suspension was first filtered through gauze and then three to four times through glass wool.…”

“…Total DNA of wild-type strain s and PaCox17::ble-47690 and -47692 strain mycelia of different ages was isolated as described elsewhere (35). Digestion with restriction enzymes, gel electrophoresis, blotting onto a nitrocellulose membrane, and hybridization with a DIGlabeled pl-intron probe (plasmid pSP17) were performed according to standard protocols (10,55,63).…”

A Podospora anserina longevity mutant with a temperature-sensitive phenotype for senescence.

“…These 10 races all undergo senescence, although at different rates (12). It has also been reported that transformation of a continuous-growth longevity mutant with the a senDNA plasmid resulted in the induction of senescence (20,22). However, this result is difficult to interpret because it was not proven that the transforming plasmid had moved into and replicated within the mitochondria.…”

Mitochondrial DNA rearrangements of Podospora anserina are under the control of the nuclear gene grisea

“…Plasmids pKP230, pKP235, pKP240, pKP245 (28), pPSB2a, pPSB2b, pPSB4 (29), and pSP17 (plDNA) (30) were used as the source to isolate cloned mtDNA fragments of the wild-type strain s. These fragments were used as mtDNAspecific probes to detect age-dependent mtDNA reorganizations.…”