Inositol-independent (inos+) revertants of Neurospora induced in inositol-requiring mutants by treatment with wild-type DNA in previous studies were found to be stable and to grow well in the absence of inositol. Genetic data presented in this paper show that a major proportion of these induced revertants rarely transmitted the inositol independence character (inos+) to their sexual progeny. Non-Mendelian transmission of the transformed character (inos+) was also found to occur in some of the sexual progeny in subsequent generations. These genetic data support the idea that the transforming DNA pieces carrying the genetic information (called exosomes) are not readily integrated into the host genome. It is suggested that elimination of most exosomes during meiosis causes a loss of the genetic information and leads to nonMendelian transmission of the induced revertant character (inos+).Since its elucidation by Avery and his coworkers (1), transformation has become a well-known process for the transfer of genetic information in bacteria. However, the study of such a genetic process in higher organisms has remained difficult due to several factors, including their diploid nature (2). Tatum (3) and Watson (4) have emphasized the significance of the use of eukaryotic microorganisms in similar studies. We have recently reinvestigated this problem in Neurospora; the results provide evidence for DNA-mediated transfer of genetic information in this eukaryotic haploid fungus (5). In the previous report we described the DNA-mediated transformation of three different genetic markers (inos-, pdx-, and rg-), each of which determines a specific defective biochemical reaction. The transformed strains were found to be stable for their DNA-induced characters during the vegetative cycle of the organism. However, some of the inositol-independent (inos+) revertants were found to be unable to transmit the DNA-induced character to their sexual progeny in ratios expected on the basis of Mendelian inheritance. These results can be explained on the basis of a rare genetic system involved in transformation of the inos-character in Neurospora. Such a genetic system has the following characteristics: (a) the transforming DNA pieces carrying the genetic information are able to replicate, transcribe, and express specific biochemical functions, and (b) in some cases these DNA pieces, called exosomes, (6), can remain unintegrated into the host genome and are subsequently liable to be eliminated during meiosis, thus leading to a non-Mendelian inheritance of the DNA-induced characters. In this paper we present genetic data which support an exosome model of transformation in Neurospora. MATERIALS AND METHODSStrains. The inositol-independent (inos+) revertant strains used in this study are those obtained during our earlier reported studies of transformation in Neurospora (5). These revertants, on the basis of their origin, belong to three groups: (a) those which originated in the control experiments without any DNA treatment, (b) those whic...

Neurospora glycogen synthetase (EC 2.4.1.11) occurs in 100,000 X g particles. The two forms (glucose-6-phosphate dependent-independent) of glycogen synthetase were solubilized and separated by digitonin treatment of the 100,000 X g particles. Glucan synthetase activity of Neurospora was found only in a cell-wall preparation. These two enzymes have been characterized in relation to the paramorphogenic action of sorbose. Sorbosegrown cultures showed a marked decrease in the specific activity of both enzymes, as compared to sucrose-grown wild-type cultures. Sorbose inhibited the activity of the wild-type enzymes both in vivo and in vitro. In the presence of 5 mM sorbose incorporation of [14C]glucose from 1'4Cluridinediphosphate glucose into glycogen by the dependent form of glycogen synthetase was completely inhibited. Thus, the paramorphogenic action of sorbose seems to result from its inhibition of these enzymes of cell-wall biosynthesis. Activities of the enzymes from the sorbose-resistant mutant, patch, were not affected by sorbose either in vivo or in vitro.In an approach to the biochemical basis of development, we have studied morphological mutants of Neurospora. Mutations that cause changes in the external appearance and growth pattern of Neurospora are known to occur at no less than 80 different genetic loci (1). About 10 of these mutations appear to affect the primary structures of particular enzymes (2-6). An alternative approach to the biochemistry of development is the study of phenocopies of genetic mutations. In this paper, we describe the result of such an approach. Tatum, Barratt, and Cutter (7) showed that irsorbose, when added to growth medium causes wild-type strains of Neurospora to grow as very restricted colonies on an agar plate, or as tight pellets in liquid medium. This discovery provided a useful methodological tool in performing high-resolution genetic analysis. However, the mechanism of the chemical induction of morphological changes by sorbose has remained essentially unknown.The morphology of Neurospora is presumably determined by its rigid cell wall. de Terra and Tatum (8) have shown that the sorbose-induced colonial morphology of Neurospora is indeed accompanied by a change in the chemical composition of the cell wall. Subsequent studies (9, 10) from this laboratory have established a marked decrease in the concentration of the cell-wall ,B-1,3-glucan in the presence of sorbose.In this paper, we describe the effect of sorbose on enzymes of polysaccharide biosynthesis. Sorbose is found to adversely affect the activities of wild-type glycogen (a-1,4-glucan) synthetase (EC 2.4.1.11) and #-1,3-glucan synthetase-both in vivo and in vitro. In the mutant patch (11), which is relatively resistant to the paramorphogenic action of sorbose, these enzymes are not affected by sorbose. MATERIALS AND METHODSStrains and Cultures. The Rockefeller University wildtype of Neurospora crassa (RL-3-8A) and the sorbose-resistant mutant, patch, described by Stadler (11) (16-to 40-hr culture) w...

Recombination junctions of several Neurospora mitochondrial DNA (mtDNA) mutants and their revertants were identified. Their nucleotide sequences and putative secondary structures were determined in order to understand the nature of the elements involved in intramolecular recombination. Multiple deletions, involving the same portion of Neurospora mtDNA, were identified in six independently isolated mutants. A 9-nucleotide repeat element, CCCCNCCCC, was found to be involved in these and other Neurospora mitochondrial recombination events. The repeat elements were clustered as hot spots on the Neurospora mtDNA and were associated with palindromic DNA sequences. The palindromes have a potential to generate hairpin structures. A much lower free energy of the putative hairpins at the 5' end of the recombination site, and the possible formation of non-B-DNA structure by polypyrimidine tracks, may be important in the initiation of recombination. Using PCR, we found low levels of a specific mitochondrial deletion in certain Neurospora mutants. Their presence in low amounts in a population with a much larger number of normal mtDNA is unexpected. Contrary to earlier belief, this finding supports the view that deleted, smaller DNA molecules are not always suppressive relative to normal mtDNAs.

We have identified a plasmid-like element within mitochondria of Neurospora crassa strain stp-B1. It is derived from the EcoRI-4 and EcoRI-6 regions of the mitochondrial DNA, and an additional 124 bp DNA segment of unknown origin. The plasmid DNA consists of an oligomeric series of circular molecules of monomer length 2.2 kbp. The abundance of the plasmid suggests its autonomous replication and the presence of an efficient origin of replication. An unusually large number of palindromes capable of forming secondary structures are present in the plasmid. Such a palindrome, located near sequences reminiscent of mammalian and fungal mtDNA origins of replication, may define the replication origin of the plasmid. This putative origin might also represent the replication origin of the wild-type mtDNA.