Transformation of Neurospora crassa with recombinant plasmids containing the cloned glutamate dehydrogenase (am) gene: evidence for autonomous replication of the transforming plasmid.

Abstract

We have characterized Neurospora crassa transformants obtained with plasmid pJR2, which consists of the Neurospora glutamate dehydrogenase (am) gene cloned in pUC8 and an am'32 host strain which contains a deletion encompassing the cloned fragment. Every one of 33 transformants tested showed extreme meiotic instability: less than 1 or 2% am+ progeny were obtained in initial or successive backcrosses between am' transformants and am'32 or in intercrosses between am+ progeny. Furthermore, am+ progeny from backcrosses gave a high proportion of auxotrophic (am) mitotic segregants during vegetative growth. These results indicate that the am+ character is not stably integrated into chromosomal DNA in any of the transformants tested. Nuclear DNAs from six transformants were analyzed by Southern hybridization. All six transformants contained sequences homologous to pJR2. Four showed restriction fragments expected for unmodified pJR2, but most showed additional bands. Southern blots of undigested DNAs showed that the plasmid sequences are present predominantly in high-molecular-weight form (larger than 20 kilobases). Southern blots showed that auxotrophic (am) progeny from a backcross to am'32 had lost restriction bands corresponding to free plasmid but retained additional bands, apparently integrated into chromosomal DNA in a nonfunctional manner. Considered together, these results are most reasonably interpreted as follows: (i) recombinant plasmids containing the am+ gene can replicate autonomously in N. crassa, (ii) the free plasmids are present in oligomeric or modified form or both, and (iii) plasmid sequences also integrate at multiple sites in the deletion host but in a nonfunctional manner. An alternate interpretation-that tandem repeats of the plasmid are integrated into chromosomal DNA but eliminated during meiosis-cannot be completely excluded. However, stable integration of the am gene can be obtained under a variety of other conditions, viz., using the am gene cloned in a phage lambda vector (J. A. Kinsey and J. A. Rambosek, Mol. Cell. Biol. 4:117-122, 1984), using derivatives of pJR2, or using pJR2 to transform a frameshift mutant.

Replicating plasmids in Schizosaccharomyces pombe: improvement of symmetric segregation by a new genetic element.

“…Recently, a report has been published that states that plasmid rearrangements also occur in S. cerevisiae (10). But it seems that rearrangements (tandem duplication, deletion) are more frequent in S. pombe, and very similar observations were made in Neurospora crassa (15). We expect that introduction of additional sequences into the vectors (e.g., for cloning experiments) may alter plasmid behavior in S.…”

DNA sequence duplications trigger gene inactivation in Neurospora crassa.

“…On the basis of these observations, Selker and colleagues (1) suggested that RIP may be a general response ofthe organism to duplications of chromosomal segments. This would explain observations of non-Mendelian inheritance of transforming DNA in Neurospora (6,7). In addition, it would explain the classical observation that segmental aneuploids are infertile ("barren") in Neurospora (see ref.…”

Nonintegrative transformation in the filamentous fungus Podospora anserina: stabilization of a linear vector by the chromosomal ends of Tetrahymena thermophila.

“…One of these sequences greatly enhances the transformation efficiency of A. nidulans, but the vector containing this ansl sequence remains integrative. In Neurospora crassa, a recombinant pUC8 plasmid carrying the am gene has been reported to be able to replicate autonomously (8). However, the transforming vector is rearranged and is present as high-molecular-weight concatemers; furthermore, some plasmid sequences are integrated into the chromosomal DNA.…”