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The expression of the am (glutamate dehydrogenase) gene is dependent upon two upstream activating sequences, designated URSam(alpha) and URSam(beta). A heteromeric nuclear protein Am Alpha Binding protein (AAB) binds specifically to a CCAAT box within the URSam(alpha) element. AAB appears to be composed of three components. We used polyclonal antiserum raised against the highly purified AAB1 subunit to isolate a partial aab-1 cDNA clone, which was then used to isolate a full-length cDNA and a genomic clone. The full-length cDNA has the potential to encode a 272 amino acid protein with a calculated molecular weight of 30 kD. Amino acid sequence obtained by Edman analysis of the AAB1 protein confirmed that the aab-1 gene had been cloned. AAB-1 shows similarity to the HAP5 protein of yeast and the CBF-C protein of rat. Each of these proteins is an essential subunit of their respective heteromeric CCAAT binding proteins. The aab1 gene maps on linkage group III of Neurospora crassa near the trp-1 locus. Disruption of the aab-1 gene results in pleiotropic effects on growth and development as well as a 50% reduction in glutamate dehydrogenase levels. Transformation of the aab-1 disruption mutant strain with the cloned genomic copy of the aab-1 gene rescued all of the phenotypic alterations associated with the aab-1 mutation.

An unstable allele of the Neurospora am (GDH) gene resulting from integration of the retrotransposon Tad3-2 into 5' noncoding sequences was found in previous work. We report that reversion to Am+ depends on DNA methylation within and upstream of Tad. Levels of methylation were correlated with the proportion of Am+ conidia, whether the cultures were derived from Am- or Am+ isolates. Reversion to Am+ did not occur when conidia were plated on 5-azacytidine, which reduces DNA methylation. The mutation dim-2, which appears to abolish DNA methylation, also prevented reversion to Am+. The native am allele, in a strain that lacked Tad elements, was replaced with am::Tad3-2 or with a deletion derivative that prevents transposition of Tad. Transformants of both classes showed instability comparable with that of the original isolates, which contain multiple Tad elements. Deletion of the upstream enhancer-like sequences, URSam alpha and beta, did not prevent the instability of am::Tad3-2. The results suggest that am expression is dependent on DNA methylation but not on proliferation or transposition of the Tad element and that the instability does not require the upstream sequences of am.

RIP (repeat-induced point mutation) efficiently mutates repeated sequences in the sexual phase of the Neurospora crassa life cycle. Nevertheless, an active LINE-like retrotransposon, Tad, was found in a N. crassa strain from Adiopodoumé. The possibility was tested that Tad might be resistant to RIP, or that the Adiopodoumé strain might be incompetent for RIP. Tad elements derived from the Adiopodoumé strain were found to be susceptible to RIP. In addition, strains lacking active Tad elements, including common laboratory strains and strains representing seven species of Neurospora, were found to have sequences closely related to Tad but with numerous mutations of the type resulting from RIP (G:C to A:T). Even the Adiopodoumé strain showed Tad-like elements with mutations characteristic of RIP. Results of crossing of an Adiopodoumé transformant with progeny of Adiopodoumé suggest that the Adiopodoumé strain is proficient at RIP. We conclude that Tad is an old transposable element that has been inactivated by RIP in most strains. Finding relics of RIP in both heterothallic and homothallic species of Neurospora implicates RIP across the genus.

New chromosome rearrangements were found in 10% or more of mitotically stable transformants. This was shown for transformations involving a variety of different markers, vectors and recipient strains. Breakpoints were randomly distributed among the seven linkage groups. Controls using untransformed protoplasts of the same strains contained almost no rearrangements. A study of molecularly characterized Am+ transformants showed that rearrangements are frequent when multiple ectopic integration events have occurred. In contrast, rearrangements are absent or infrequent when only the resident locus is restored to am+ by a homologous event. Sequences of the transforming vector were genetically linked to breakpoints in 6 of 10 translocations that were examined using Southern hybridization or colony blots.

We have analyzed the junctions involved in two examples of ectopic integration of plasmids containing the am+ (glutamate dehydrogenase) gene into a strain of Neurospora crassa bearing a complete deletion of the am locus. In one transformed strain a single copy of plasmid DNA had been integrated into linkage group (LG) III DNA without the loss of chromosomal DNA. In contrast, 450 bp had been lost from plasmid sequences at the site of integration. The transforming DNA used was circular, so we postulate that the plasmid was linearized and truncated prior to its integration by end joining into a break in LG III DNA. There was no significant homology between the incoming DNA and DNA at the site of integration. The second transformed strain resulted from transformation with a linearized plasmid. It contained multiple integrated copies of plasmid DNA, one of which was recloned, together with adjacent chromosomal DNA, by plasmid rescue in Escherichia coli. Prior to integration into chromosomal DNA, the linear plasmid had been truncated by 64 bp on one end and 3.2 kbp on the other end. One end of the integrated DNA was adjacent to DNA from the right arm of LG I, while the other end was integrated into a copy of a repetitive sequence. Restriction fragment length polymerism mapping showed that integration was in a copy of the repetitive sequence that is linked to the previously unassigned telomere M11 and is distantly linked to the LG VI marker con-11. Genetic analysis revealed that a long segment of LG I containing all markers from un-1 to the right tip has been translocated to the right end of LG VI. Tetrad analysis showed that the integrated DNA was closely linked to the translocation. We conclude that the transforming DNA was truncated and joined to DNA from two different chromosomes by end joining during the formation of a quasiterminal translocation, T(IR----VIR) UK-T12. We also conclude that the previously unassigned telomere, M11, is the right end of LG VI.

The process designated RIP (repeat-induced point mutation) alters duplicated DNA sequences in the sexual cycle of Neurospora crassa. We tested whether non-Neurospora sequences are susceptible to RIP, explored the basis for the observed immunity to this process of a diverged tandem duplication that probably arose by a natural duplication followed by RIP (the Neurospora zeta-eta region), and investigated whether RIP extends at all into unique sequences bordering a duplicated region. Bacterial sequences of the plasmid pUC8 and of a gene conferring resistance to hygromycin B were sensitive to RIP in N. crassa when repeated in the genome. When the entire 1.6-kb zeta-eta region was duplicated, it was susceptible to RIP, but was affected by it to a lesser extent than other duplications. Only three of 62 progeny from crosses harboring unlinked duplications of the region showed evidence of changes. We attribute the low level of alterations to depletion of mutable sites. The stability of the zeta-eta region in strains having single copies of the region suggests that the 14% divergence of the tandem elements is sufficient to prevent RIP. DNA sequence analysis of unduplicated pUC8 sequences adjacent to a duplication revealed that RIP continued at least 180 bp beyond the boundary of the duplication. Three mutations occurred in the 200-bp segment of bordering sequences examined.

The Tad transposon of Neurospora crassa appears to be a LINE-like element with very restricted distribution within the genus Neurospora. When forced heterokaryons were constructed between strains which did and did not contain Tad, the nuclei of the naive nuclear type rapidly acquired Tad elements. The elements acquired by naive nuclei are active, since they can pass Tad to other naive nuclei in subsequent heterokaryons. When heterokaryons are passaged by serial transfer, the load of acquired Tad elements appears to increase, indicating that transposition is continuing in these heterokaryons, even after all of the naive nuclei have acquired Tad. In normal heterokaryons of Neurospora, nuclei do not fuse. An experiment to test for the possibility that Tad promotes nuclear fusion gave negative results. Thus Tad appears to have a cytoplasmic intermediate in its transposition. When heterokaryon incompatible strains were cocultured, there was no indication that Tad elements could be transferred to the naive strain, suggesting that Tad is not a virus. These data are consistent with the transposition of Tad via RNA and cDNA intermediates, as has been postulated to occur with LINE-like elements.

Utilizing a combination of flanking marker analysis and deletion mapping we have constructed a fine structure map of the am locus which includes 63 point mutants and ten unique deletions. Positions of point mutants can be rapidly assigned to one of 13 segments within the gene on the basis of crosses to nine deletion strains.

The mutant strain am126 was isolated, using the direct selection procedure, after nitrous acid mutagenesis. It produced neither measurable NADP-dependent glutamate dehydrogenase (GDH) nor immunologically cross-reacting material. That the am126 strain produced some form of GDH product was shown by the fact that it complemented several other am mutant strains. The GDH formed by complementation between am126 and each of two other am mutants was relatively thermolabile, but could not be distinguished from wild-type GDH formed by electrophoresis in polyacrylamide gels. This, together with the relatively high yield of the complementation enzymes, suggest that the am126 product is a polypeptide chain not grossly abnormal in structure. The spontaneous revertant frequency was between 0.3 and 3 prototrophic revertants per 10(5) live cells. This frequency was at least 40 times greater than that for am19, which had the second highest spontaneous revertant frequency among the mutants tested. Neither meiosis nor mutagenesis increased the revertant frequency, nor did incubation at elevated temperatures lower it. Sixty-eight revertant strains were examined for thermostability of their GHD. All appeared to be identical to wild type. Seven of the revertant strains were also tested for instability with regard to forward mutation to am auxtrophy. None was found to be unstable. Models for the genetic instability of the am126 mutation are discussed.

Forty-eight new mutations at the am locus of Neurospora crassa have been characterized. Nineteen mutations were induced by UV; of these, eight were missense, two were frameshifts, two were nonsense, three were deletions and four were unidentified. Twenty-nine mutations were induced with nitrous acid; of these, twenty-one were missense, three were frameshifts, one was nonsense, two were deletions and one was genetically unstable.

The am locus of Neurospora codes for NADP-dependent glutamate dehydrogenase (GDH). Four new am mutants that produced mutationally altered GDH have been characterized. Mutant am119 is a CRM-negative, complementing mutant that maps between am2 and am1. The other three mutants are CRM formers that produce varieties of GDH that can be activated by glutamate or succinate. The GDH of am130 and am131 is similar in terms of activation properties to that of am3. The GDH of am122 requires very high concentrations of dicarboxylate for activity. The mutation in am130 maps between am14 and am2 and resulted in a replacement at residue 75 of the GDH (pro leads to ser). The mutation in am122 maps near am11 and apparently resulted in the replacement of the tryptophan residue at position 389 with an unknown amino acid. The mutation in am131 maps between am2 and am1.