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Tad is a LINE-like retrotransposon of the filamentous fungus Neurospora crassa. We have analyzed both expression and transposition of this element using strains with a single copy of Tad located in the 5' noncoding sequences of the am (glutamate dehydrogenase) gene. Tad in this position has been shown to carry a de novo cytosine methylation signal which causes reversible methylation of both Tad and am upstream sequences. Here we find that methylation of the Tad sequences inhibits both Tad expression and transposition. This inhibition can be relieved by the use of 5-azacytidine, a drug which reduces cytosine methylation, or by placing the Tad/am sequences in a dim-2 genetic background.

Tad is an active LINE-like retrotransposon isolated from the Adiopodoumé strain of Neurospora crassa. Extensive analysis of other Neurospora strains has revealed no other strain with active Tad, but all strains tested have multiple copies of defective Tad elements. We have examined the ability of Tad to survive during the sexual cycle of Neurospora and find that active Tad is rapidly eliminated. The characteristics of this elimination suggest that the repeat-induced point mutation (RIP) mechanism was responsible. By the use of transformation to switch the mating type of the Adiopodoumé strain we concluded that this strain is not defective in the RIP process. Analysis of defective Tad elements isolated from a variety of strains indicates that the major difference between these elements and active Tad is due to the presence of a large number of G-C to A-T transition mutations. This would be expected if the changes were due primarily to the RIP process. Mapping of a selection of defective Tad elements reveals that they are present on all of the chromosomes; however, many of the elements are not widely shared among strains. This suggests that repeated introduction and elimination of Tad elements has occurred. Mechanisms that might be responsible for this repeated introduction are discussed., (Copyright 2001 Academic Press.)

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.

Expression of the Neurospora crassa am (NADP-specific glutamate dehydrogenase) gene is controlled by two upstream enhancer-like elements designated URSam alpha and URSam beta. URSam alpha is localized between - 1.3 and - 1.4 kb with respect to the major transcriptional start site. Deletion of a 90 bp sequence containing this element resulted in the loss of approximately 50% of normal glutamate dehydrogenase expression. Gel mobility shift analysis indicated that a nuclear protein from Neurospora binds in a specific manner to sequences within the 90 bp fragment. We have now used a combination of ion-exchange and affinity chromatography to purify this nuclear protein, which we call Am Alpha Binding protein (AAB). The activity was monitored by gel shift analysis. The protein was purified more than 14,000-fold with a yield of approximately 7%. The purified protein appears as a heteromer on denaturing polyacrylamide gel electrophoresis, with only two strong bands visible in silver-stained preparations. One band has an apparent molecular mass of 40 kDa, the other appears as a doublet with an apparent molecular mass of 30 kDa. DNAse I protection analysis indicated a protected region consisting of 30 bp, which contains a CCAAT pentanucleotide motif. Mutagenesis of the CCAAT motif abolished the binding of AAB to the DNA fragment.

Tad is a LINE-like retrotransposon of Neurospora crassa. The element was originally detected and cloned using the am gene as a transposon trap in hybrid strains derived from a cross of Adiopodoume (a wild collected strain) and a laboratory strain devoid of Tad elements. We report the cloning and sequencing of an active Tad element, Tad1-1, which is capable of independent transposition. Transposition was demonstrated by screening for transfer of the element from a donor nucleus that contained the Tad1-1 element as the only active Tad, into a naive nucleus within a forced heterokaryon. We also report here the sequence analysis of Tad1-1, and its comparison with the sequence of another active element, Tad3-2. These elements are approximately 7 kb in length. They contain two long open reading frames (ORFs) encoded on the strand of the same polarity as the full-length transcript. ORF1 encodes a putative protein of 486 amino acids. Homology to the first ORF of other LINE elements is confined to three cysteine-rich motifs, located near the carboxy-terminus, that are thought to be involved in binding nucleic acids. The second ORF is 1156 amino acids in length and shows homology to the reverse transcriptase domains of various retroviruses and retrotransposons. Tad1-1 and Tad3-2 differ in only ten positions over their whole length.

We have used gel mobility shift assays to scan 1.7 kb of 5' non-coding sequence of the am (glutamate dehydrogenase) gene of Neurospora crassa for binding by partially fractionated Neurospora proteins. Using genetic analysis this region had been shown to play an important role in the control of glutamate dehydrogenase (GDH) expression. Gel mobility shift analysis identified three regions to which Neurospora proteins bind specifically. Two of these corresponded to the two elements previously defined by genetic analysis (URSam alpha and URSam beta). The third protein binding site appears to be unrelated to am gene expression. Competition experiments showed that the proteins that bind to the URSam alpha and URSam beta elements are different. The URSam alpha element was shown to contain two independent binding sites for the URSam alpha binding protein(s). Both fragments contain a CCAAT motif, suggesting that URSam alpha binding protein(s) may be members of one of the CCAAT-binding protein families. The effect of deletion of either the URSam alpha or URSam beta elements on catabolite induction of am expression was also determined. Both elements appear to act as constitutive enhancers of gene expression.

We have constructed a series of deletions in the 5' non-coding sequences of the cloned Neurospora crassa am gene which specifies NADP specific glutamate dehydrogenase. All of the deletions begin at -4.4 kb with respect to the am transcription start site and extend for various distances toward the am gene. Using vectors with a truncated fragment of the am gene, we introduced these deletions into the chromosome upstream of am by transformation. Analysis of glutamate dehydrogenase expression in strains with the deletion mutations confirmed that there are two upstream regulatory sequences (URS) that control the expression of the am gene. The more distal of these elements (URSam beta) has been limited to the 157 bp between -1924 and -2081 with respect to the start of am transcription. The proximal element (URSam alpha) was limited to the 97 bp between -1296 and -1393. The DNA sequence of the entire region was determined. Within the sequences that contain the URS elements several regions of homology with yeast UAS sequences were found. Gel mobility assays with DNA fragments containing the URS elements indicated that sequences in both elements are bound by nuclear proteins from Neurospora. The interaction of these proteins and the DNA fragments was found to be specific.

We used lambda and plasmid vectors containing the am+ gene in an insert of from 2.7 to 9.1 kb, to transform am point mutant and deletion strains. A total of 199 transformants were examined with the potential to yield am+ transformants by homologous recombination. When we used vectors that had 9.1 kb of homology with the chromosomal DNA, 30% of the transformants obtained were the result of homologous recombination regardless of whether the vector was a lambda molecule, a circular plasmid, or a plasmid that had been linearized prior to transformation. When vectors with up to 5.1 kb of homology were used, very few transformants (1 of 89 tested) resulted from homologous recombination. Of a sample of 29 ectopic integration events obtained by transformation with the 9.1 kb fragment cloned in a lambda vector, 18 included a major part (usually almost all) of both arms of lambda with the entire Neurospora 9.1 kb insert between them. Four included only lambda long arm sequence together with an adjacent segment of the insert containing the am gene. The remaining seven were the result of multiple integrations. There was no evidence of circularization of the lambda vector prior to integration. All transformants that had multiple copies of the am gene appeared to be subject to the RIP process, which causes multiple mutations in duplicated sequences during the sexual cycle.

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.

In a previous study the alteration in the amino acid sequence of Neurospora crassa NADP-specific glutamate dehydrogenase (GDH) resulting from two mutually compensating frameshift mutations was used to deduce the first 17 nucleotides of the coding sequence of the am gene. In the work reported here, a synthetic 17-mer corresponding to the deduced sequence was shown to hybridize strongly to a 9-kb HindIII fragment from N. crassa wild-type DNA but not to any corresponding fragment from the DNA of a mutant strain known to be deleted for most or all of the gene. Wild-type HindIII fragments were fractionated for size and a fraction centering around 9 kb was cloned in vector lambda L47. Two clones carrying the strongly hybridizing fragment were identified. The hybridization to the 17-mer was localized within a 2.7-kb BamHI fragment and, within this, to a 700-bp BamHI-Bg/II subfragment. 5' end-labelled polyadenylated RNA isolated from wild-type mycelium hybridized to the 2.7-kb BamHI fragment and not appreciably to flanking fragments. The partial sequence analysis of the BamHI-Bg/II fragment has confirmed that the 17-mer probe matches the coding sequence at the 5' end of the gene and has also revealed an intervening sequence 67 bp in length, interrupting codon 15. Both the 9-kb HindIII fragment and the 2.7-kb BamHI fragment have been shown to be capable of transforming the deletion mutant to prototrophy and ability to produce GDH. Analysis of one transformant showed that the am gene was integrated, together with a part of the long arm of the lambda vector, at an unusual locus. This transformant, in which the am gene does not show its normal linkage to the linkage group 5 marker inl, was found to produce GDH to about 20% of the normal level.

We have cloned the unstable am mutant gene, am126, as well as the am gene from an am126 revertant. The mutation is a result of a 33-bp duplication that repeats a sequence starting 13 bp upstream of the 3' splice junction between intron 1 and exon 2 and extends 20 bp into exon 2. In addition, there is a G----A transition 2 bp upstream of the first copy of the duplicated sequence. In the revertant gene the wild-type sequence is precisely recovered, involving both the loss of the duplication and a reversion (A----G) of the associated transition. Our data suggest that only the more 5' of the two 3' splice junctions present in the duplicated version of the gene is used. This favors a 5'----3' scanning mechanism for exon splicing.

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.

A simple colony blot procedure was used to screen 336 Neurospora strains for the presence of the transposable element Tad. These strains included the standard laboratory wild types, all of the available Neurospora isolates collected from the Ivory Coast, and all wild-collected Neurospora crassa isolates available from the Fungal Genetics Stock Center. Tad was found only in the strain of origin from Adiopodumé, Ivory Coast, where it is present in multiple copies. Three other strains of African origin were found to have single copy sequences that are related to Tad.

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.

During the germination and growth of Neurospora conidia, amino acid permease systems I (neutral) and II (general) increase in specific activity. System III (basic) decreases in specific activity with the onset of germination. System I shows two peaks of activity during the logarithmic phase of growth. One peak occurs at 6 h, the other at 12 h of growth. Both peaks are abolished in the mtr mutant. Both peaks have a Km for phenylalanine of 40 muM. The peaks of system I activity appear to correlate with morphological changes.

Specific in vitro-generated insertion, replacement, and deletion mutations have been integrated near the chromosomal locus of am (NADP-specific glutamate dehydrogenase) of Neurospora crassa. Two approaches have been successful. One approach used am+-containing vectors capable of integrating at any site in the genome. This technique was used to introduce a specific 700 bp insertion near the am locus and to replace chromosomal sequences near am with plasmid DNA. Efficiency was low, however, and many transformants had to be screened to find the desired alterations among the ectopic insertions unless the incoming DNA had a large region of homology with the am region. A second approach increased the efficiency by using vectors containing a truncated am gene, so that prototrophs could arise only by homologous recombination. Overall transformation frequency was reduced relative to the first method, but a large fraction of the transformations involved specific alterations of the am region.

An 8-month-old infant boy with median facial cleft syndrome had eyelid coloboma, symblepharon, and a cytic mass in the left upper eyelid. The mass proved to be an ectatic cornea containing a large cystic lens. Maldevelopment of the entire anterior segment of the eye was also present, although the posterior globe was well formed. We postulate that an area of localized abnormal mesodermal differentiation and fusion at the 17- to 20-mm stage of development served as a common mechanism for all the defects noted.

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.

A tryptophan auxotroph of Neurospora crassa, trp-5, has been characterized as a mutant with a deficient tryptophanyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.2) activity. When assayed by tryptophanyl-tRNA formation, extracts of the mutant have less than 5% of the wild-type specific activity. The adenosine triphosphate-pyrophosphate exchange activity is at about half the normal level. In the mutant derepressed levels of anthranilate synthetase and tryptophan synthetase were associated with free tryptophan pools equal to or higher than those found in the wild type. We conclude that a product of the normal tryptophanyl-tRNA synthetase, probably tryptophanyl-tRNA, rather than free tryptophan, participates in the repression of the tryptophan biosynthetic enzymes.

A new p-fluorophenylalanine resistant mutant of Neurospora (fpr-1) was isolated. It is unaffected by the suppressor of a previously described resistance mutant, mtr. The fpr-1 locus is on linkage group V, tightly linked to act-2. The expression of resistance to p-fluorophenylalanine by fpr-1 can be suppressed by genes controlling a requirement for lysine or arginine. The suppression seems to involve an increased sensitivity of the lysine and arginine auxotrophs to p-fluorophenylalanine.