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We have characterized Neurospora crassa transformants obtained with plasmid pJR2, which consists of the Neurospora glutamate dehydrogenase (am) gene cloned in pUC8 and an am132 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 am132 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 am132 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: recombinant plasmids containing the am+ gene can replicate autonomously in N. crassa, the free plasmids are present in oligomeric or modified form or both, and 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.

We used DNA containing the am gene of Neurospora crassa, cloned in the lambda replacement vector lambdaL-47 (this clone is designated lambdaC-10), and plasmid vector subclones of this DNA to transform am deletion and point mutant strains. By means of subcloning, all sequences required for transformation to am prototrophy and expression of glutamate dehydrogenase have been shown to reside on a 2.5-kilobase BamHI fragment. We also characterized several am+ strains that were obtained after transformation with lambdaC-10. These strains showed Mendelian segregation of the am+ gene, although less than 50% of the transformed strains showed the normal linkage relationship of am with inl. In all cases tested, the strains had incorporated lambda DNA as well. The lambda DNA also showed a Mendelian segregation pattern. In one case, the incorporation of am DNA in a novel position was associated with a mutagenic event producing a strain with a very tight colonial morphology. In all cases in which the am+ gene had become the resident of a new chromosome, glutamate dehydrogenase was produced to only 10 to 20% of the wild-type levels.