Premeiotic Change of Nucleolus Organizer Size in Neurospora

We have investigated the heritability of nucleolus organizer region (NOR) size in Neurospora crassa. By pulsed-field gel electrophoresis, we followed in genetic crosses the size of the normal or "terminal" NORs and the size of a small interstitial NOR. Tetrad analysis revealed that changes in NOR size occur frequently in the sexual phase. Moreover, most size changes occurred in the period between fertilization and meiosis, although some changes occurred during and after meiosis. Unexpectedly, increases and decreases in NOR size were not equally frequent: decreases were more common. The NOR size changes generated during meiosis were not the result of unequal crossing over between NORs on homologous chromosomes.     

Ribosomal DNA Is a Site of Chromosome Breakage in Aneuploid Strains of Neurospora

In wild-type strains of Neurospora crassa, the rDNA is located at a single site in the genome called the nucleolus organizer region (NOR), which forms a terminal segment on linkage group (LG) V. In the quasiterminal translocation strain T(I;V)AR190, most of the right arm of LG I moved to the distal tip of the NOR, and one or a few rDNA repeat units are moved to the truncated right arm of LG I. I report here that, in partial diploid strains derived from T(I;V)AR190, large terminal deletions result from chromosome breakage in the NOR. In most of these partial diploids, chromosome breakage is apparently frequent and the breakpoints occur in many parts of the NOR. The rDNA ends resulting from chromosome breakage are "healed" by the addition of new telomeres. Significantly, the presence of ectopic rDNA creates a new site of chromosome breakage in the genome of partial diploids. These results raise the possibility that, under certain conditions, rDNA is a region of fragility in eukaryotic chromosomes.     

Analysis of two additional loci in Neurospora crassa related to Spore killer-2

Two new loci found in one strain of Neurospora crassa (P2604) collected in Malaya are related to the meiotic drive system Spore killer Sk-2. Sk-2 was found in Neurospora intermedia and introgressed into N. crassa. P2604 showed high resistance to killing when crossed to Sk-2. This resistance was found to be linked to, but not allelic to, resistance locus r(Sk-2) on LGIIIL. Analysis showed that the high resistance phenotype of P2604 requires resistance alleles at two different loci on LGIIIR. Strains carrying a resistance allele at only the proximal or the distal locus, respectively, were obtained and intercrossed. Highly resistant strains were obtained by rejoining the two genes. The proximal locus alone confers a low level of resistance. This locus was named pr(Sk-2) for partial resistance to Sk-2. The distal locus was named mod(pr) because its only known phenotype is to modify pr(Sk-2).     

Chromosome Rearrangements That Involve the Nucleolus Organizer Region in Neurospora

In ~3% of Neurospora crassa rearrangements, part of a chromosome arm becomes attached to the nucleolus organizer region (NOR) at one end of chromosome 2 (linkage group V). Investigations with one inversion and nine translocations of this type are reported here. They appear genetically to be nonreciprocal and terminal. When a rearrangement is heterozygous, about one-third of viable progeny are segmental aneuploids with the translocated segment present in two copies, one in normal position and one associated with the NOR. Duplications from many of the rearrangements are highly unstable, breaking down by loss of the NOR-attached segment to restore normal chromosome sequence. When most of the rearrangements are homozygous, attenuated strands can be seen extending through the unstained nucleolus at pachytene, joining the translocated distal segment to the remainder of chromosome 2. Although the rearrangements appear genetically to be nonreciprocal, molecular evidence shows that at least several of them are physically reciprocal, with a block of rDNA repeats translocated away from the NOR. Evidence that NOR-associated breakpoints are nonterminal is also provided by intercrosses between pairs of translocations that transfer different-length segments of the same donor-chromosome arm to the NOR.     

Structural variations and optional introns in the mitochondrial DNAs of Neurospora strains isolated from nature

Mitochondrial DNAs from ten wild-type Neurospora crassa, Neurospora intermedia, and Neurospora sitophila strains collected from different geographical areas were screened for structural variations by restriction enzyme analysis. The different mtDNAs show much greater structural diversity, both within and among species, than had been apparent from previous studies of mtDNA from laboratory N. crassa strains. The mtDNAs range in size from 60 to 73 kb, and both the smallest and largest mtDNAs are found in N. crassa strains. In addition, four strains contain intramitochondrial plasmid DNAs that do not hybridize with the standard mtDNA. All of the mtDNA species have a basically similar organization. A 25-kb region that includes the rRNA genes and most tRNA genes shows very strong conservation of restriction sites in all strains. The 2.3-kb intron found in the large rRNA gene in standard N. crassa mtDNAs is present in all strains examined, including N. intermedia and N. sitophila strains. The size differences between the different mtDNAs are due to insertions or deletions that occur outside of the rRNA-tRNA region. Restriction enzyme and heteroduplex mapping suggest that four of these insertions are optional introns in the gene encoding cytochrome oxidase subunit I. Mitochondrial DNAs from different wild-type strains contain zero, one, three, or four of these introns.     

A relationship between carotenoid accumulation and the distribution of species of the fungus Neurospora in Spain

The ascomycete fungus Neurospora is present in many parts of the world, in particular in tropical and subtropical areas, where it is found growing on recently burned vegetation. We have sampled the Neurospora population across Spain. The sampling sites were located in the region of Galicia (northwestern corner of the Iberian peninsula), the province of Cáceres, the city of Seville, and the two major islands of the Canary Islands archipelago (Tenerife and Gran Canaria, west coast of Africa). The sites covered a latitude interval between 27.88° and 42.74°. We have identified wild-type strains of N. discreta, N. tetrasperma, N. crassa, and N. sitophila and the frequency of each species varied from site to site. It has been shown that after exposure to light Neurospora accumulates the orange carotenoid neurosporaxanthin, presumably for protection from UV radiation. We have found that each Neurospora species accumulates a different amount of carotenoids after exposure to light, but these differences did not correlate with the expression of the carotenogenic genes al-1 or al-2. The accumulation of carotenoids in Neurospora shows a correlation with latitude, as Neurospora strains isolated from lower latitudes accumulate more carotenoids than strains isolated from higher latitudes. Since regions of low latitude receive high UV irradiation we propose that the increased carotenoid accumulation may protect Neurospora from high UV exposure. In support of this hypothesis, we have found that N. crassa, the species that accumulates more carotenoids, is more resistant to UV radiation than N. discreta or N. tetrasperma. The photoprotection provided by carotenoids and the capability to accumulate different amounts of carotenoids may be responsible, at least in part, for the distribution of Neurospora species that we have observed across a range of latitudes.     

Reversal of a Neurospora Translocation by Crossing over Involving Displaced Rdna, and Methylation of the Rdna Segments That Result from Recombination

In translocation OY321 of Neurospora crassa, the nucleolus organizer is divided into two segments, a proximal portion located interstitially in one interchange chromosome, and a distal portion now located terminally on another chromosome, linkage group I. In crosses of Translocation X Translocation, exceptional progeny are recovered nonselectively in which the chromosome sequence has apparently reverted to Normal. Genetic, cytological, and molecular evidence indicates that reversion is the result of meiotic crossing over between homologous displaced rDNA repeats. Marker linkages are wild type in these exceptional progeny. They differ from wild type, however, in retaining an interstitial block of rRNA genes which can be demonstrated cytologically by the presence of a second, small interstitial nucleolus and genetically by linkage of an rDNA restriction site polymorphism to the mating-type locus in linkage group I. The interstitial rDNA is more highly methylated than the terminal rDNA. The mechanism by which methylation enzymes distinguish between interstitial rDNA and terminal rDNA is unknown. Some hypotheses are considered.     

Molecular Cloning of a Gene (Cfp) Encoding the Cytoplasmic Filament Protein P59nc and Its Genetic Relationship to the Snowflake Locus of Neurospora Crassa

P59Nc is a 59-kD polypeptide associated with 8-10-nm diameter cellular filaments in normal Neurospora crassa strains. Abnormally sized and shaped bundles of these structures are present in N. crassa strains carrying mutations at the locus sn (snowflake). By using molecular cloning and restriction fragment length polymorphism (RFLP) segregation analysis strategies we show here that sn is not the genetic locus of P59Nc. Several P59Nc cDNAs were cloned from a N. crassa lambda GT11 library after immunoscreening with specific polyclonal anti-P59Nc antibodies. Additional longer cDNAs were obtained from a N. crassa cDNA-lambda ZAP library. When used as probes in Southern blots of total DNA from wild-type strains, multicent-2 (a multiple mutant strain), and snowflake mutants, the P59Nc cDNAs revealed comparable patterns of hybridizing bands for all of the restriction enzymes tested. Analysis of segregation of BclI and ClaI RFLPs, detected in the genomic region of the P59Nc gene (locus cfp: cellular filament polypeptide), among a set of strains designed for RFLP mapping, or among selected progeny of crosses involving a snowflake parent, respectively, indicate that (i) there is in N. crassa a single cfp locus positioned on the right arm of linkage group VII between the locus for and the proximal breakpoint of the translocation T(VII----I)5936; (ii) the sn mutations in the centromere region of chromosome I do not represent translocations of cfp; and (iii) the snowflake mutants possesses a normal copy of the P59Nc gene on their chromosomes VII.(ABSTRACT TRUNCATED AT 250 WORDS)     

Signal for DNA methylation associated with tandem duplication in Neurospora crassa.

Most cytosine residues are subject to methylation in the zeta-eta (zeta-eta) region of Neurospora crassa. The region consists of a tandem direct duplication of a 0.8-kilobase-pair element including a 5S rRNA gene. The repeated elements have diverged about 15% by the occurrence of numerous CG to TA mutations, which probably resulted from deamination of methylated cytosines. Most but not all common laboratory strains of N. crassa have methylated duplicated DNA at the zeta-eta locus. However, many strains of N. crassa and strains of N. tetrasperma, N. sitophila, and N. intermedia have one instead of two copies of the homologous DNA and it is not methylated. A cross of strains differing at the zeta-eta locus produced progeny which all had duplicated, methylated, or unique, unmethylated DNA, like the parental strains. We conclude that a signal causing unprecedented heavy DNA methylation is present in the zeta-eta region.     

Optimized vectors and selection for transformation of Neurospora crassa and Aspergillus nidulans to bleomycin and phleomycin resistance.

To provide a dominant selectable marker for transformation of Neurospora crassa strains lacking specific auxotrophic mutations, we have engineered the bleomycin (Bm) resistance-encoding gene (ble) from the bacterial transposon Tn5 for expression in N. crassa. The coding region of the ble gene was fused to the promoter and terminator regions of the N. crassa am gene. In some vectors, multiple cloning sites were placed flanking the ble gene to provide a versatile ble cassette. When introduced into N. crassa, the hybrid ble gene conferred resistance to greater than 15 micrograms Bm/ml. Under optimal conditions, the levels of Bm required (2.5 micrograms/ml) make even large-scale transformation experiments very economical. Aspergillus nidulans could also be efficiently transformed to Bm resistance using the N. crassa ble gene fusion. Since the ble gene functions in both N. crassa and A. nidulans, the gene should be useful as a transformation marker for the many other filamentous fungi which are sensitive to Bm.     

Cloning of methylated transforming DNA from Neurospora crassa in Escherichia coli.

An arg-2 mutant of Neurospora crassa was transformed to prototrophy with a pBR322-N. crassa genomic DNA library. Repeated attempts to recover the integrated transforming DNA or segments thereof by digestion, ligation, and transformation of Escherichia coli, with selection for the plasmid marker ampicillin resistance, were unsuccessful. Analyses of a N. crassa transformant demonstrated that the introduced DNA was heavily methylated at cytosine residues. This methylation was shown to be responsible for our inability to recover transformants in standard strains of E. coli; transformants were readily obtained in a strain which is deficient in the two methylcytosine restriction systems. Restriction of methylated DNA in E. coli may explain the general failure to recover vector or transforming sequences from N. crassa transformants.     

Molecular comparison of the negative-acting nitrogen control gene,nmr, inNeurospora crassa and otherNeurospora and fungal species

In Neurospora crassa, the expression of unlinked structural genes which encode nitrogen catabolic enzymes is subject to genetic and metabolic regulation. The negative-acting nmr regulatory gene appears to play a role in nitrogen catabolite repression. Using the N. crassa nmr gene as a probe, homologous sequences were identified in a variety of other filamentous fungi. The polymerase chain reaction was used to isolate the nmr-like gene from the exotic Mauriceville strain of N. crassa and from the two related species, N. intermedia and N. sitophila. Sequence comparisons were carried out with a 1.7-kb DNA segment which includes the entire coding region of nmr plus 5' and 3' noncoding sequences. The size of the nmr coding region was identical in all three Neurospora species. Approximately 30 nucleotide base substitutions were found in the coding region of the nmr gene of each of the sister species when compared to the standard N. crassa sequence. However, most of the base changes occurred in third codon positions and were silent. The NMR proteins of N. sitophila and of N. intermedia display only three and four amino acid substitutions, respectively, from the N. crassa protein. Two regions of high variability, which include deletions and insertions of bases, were found in the 5' and 3' noncoding regions of the gene.     

Molecular analysis of mutants of the <Emphasis Type="Italic">Neurospora</Emphasis> adenylosuccinate synthetase locus

The ad-8 gene of Neurospora crassa, in addition to being used for the study of purine biology, has been extensively studied as a model for gene structure, mutagenesis and intralocus recombination. Because of this there is an extensive collection of well-characterized N. crassa ad-8 mutants in the Fungal Genetics Stock Center collection. Among these are spontaneous mutants and mutants induced with X-ray, UV or chemical mutagens. The specific lesions in these mutants have been genetically mapped at high resolution. We have sequenced the ad-8 locus from 13 of these mutants and identified the molecular nature of the mutation in each strain. We compare the historical fine-structure map to the DNA and amino acid sequence of each allele. The placement of the individual lesions in the fine-structure map was more accurate at the 5' end of the gene and no mutants were identified in the 3' untranslated region of this gene. We additionally analysed ad-8(+) alleles in 18 N. crassa strains subjected to whole-genome sequence analysis and describe the variability among Neurospora strains and among fungi and other organisms.     

Amplification of the nucleolus organizer region during the sexual phase of Neurospora crassa

Previously we have shown that the nucleolus organizer region (NOR) of Neurospora crassa displays frequent size changes during crosses. In these initial studies, we observed that decreases in NOR size are far more common than increases. Here, we have investigated the inheritance of NOR size in a strain with an unusually small NOR. We call this strain SNO for small nuclelous organizer. We found that progeny that inherit their rDNA from SNO receive either an NOR that is larger than that of SNO or, rarely, the same size, but never an NOR that is smaller than that of SNO. The number of progeny that inherit their NOR from SNO is not significantly different from the number that inherit their NOR from the other parent in the cross. This argues against the idea that the failure to find progeny with NORs smaller than that of SNO is due to inviability of spores carrying such an NOR, or that it is due to unconscious bias by the experimenter against isolating such spores. These results can most easily be explained by a combination of unequal sister chromatid exchanges in the rDNA, or sister chromatid conversion, coupled with selection againts nuclei harboring small NORs during the premeiotic phase of the Neurospora life cycle. Other, less conventional, explanations are also possible, such as directed increase in the target NOR without corresponding loss at some other NOR.     

High-density detection of restriction-site-associated DNA markers for rapid mapping of mutated loci in Neurospora

The wealth of sequence information available for Neurospora crassa and other fungi has greatly facilitated evolutionary and molecular analyses of this group. Although "reverse" genetics, in which genes are first identified by their sequence rather than by their mutant phenotypes, serves as a valuable new approach for elucidating biological processes, classical "forward" genetic analysis is still extremely useful. Unfortunately, mapping mutations and identifying the corresponding genes has typically been slow and laborious. To facilitate forward genetics in Neurospora, we have adapted microarray-based restriction-site-associated DNA (RAD) mapping for use with N. crassa oligonucleotide microarrays. This technique was used to simultaneously detect an unprecedented number of genomewide restriction site polymorphisms from two N. crassa strains: Mauriceville and Oak Ridge. Furthermore, RAD mapping was used to quickly map a previously unknown gene, defective in methylation-7 (dim-7).     

Specificity of Repeat-Induced Point Mutation (Rip) in Neurospora: Sensitivity of Non-Neurospora Sequences, a Natural Diverged Tandem Duplication, and Unique DNA Adjacent to a Duplicated Region

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.     

Nucleotide sequence of the Neurospora crassa trp-3 gene encoding tryptophan synthetase and comparison of the trp-3 polypeptide with its homologs in Saccharomyces cerevisiae and Escherichia coli

The complete nucleotide sequence of the Neurospora crassa trp-3 gene-encoding tryptophan synthetase has been determined; we present an analysis of its structure. A comparison of the deduced amino acid sequence of the trp-3 polypeptide with its homologs in Saccharomyces cerevisiae (encoded by the TRP5 gene) and Escherichia coli (encoded by the trpA and trpB genes) shows that the A and B domains (amino acid segments homologous to the trpA and trpB polypeptides, respectively) of the N. crassa and yeast polypeptides are in the same order (NH2-A-B-COOH). This arrangement is the reverse of the gene order characteristic of all prokaryotes that have been examined. N. crassa tryptophan synthetase has strong homology to the yeast TRP5 polypeptide (A domains have 54% identity; B domains have 75% identity), and somewhat weaker homology to the E. coli trpA and trpB polypeptides (A domains have 31% identity; B domains have 50% identity). The two domains of the N. crassa polypeptide are linked by a connector of 54-amino acid residues that has less than 25% identity to the 45-residue connector of the yeast polypeptide, although secondary structure analysis predicts both connectors would be alpha-helical. In contrast to the yeast TRP5 gene, which has no introns, the trp-3 coding region is interrupted by two introns 77 and 71 nucleotides in length. Both introns are located near the 5'-end of the gene and therefore not near the segment encoding the connector.     

The homologue of het-c of Neurospora crassa lacks vegetative compatibility function in Fusarium proliferatum

For two fungal strains to be vegetatively compatible and capable of forming a stable vegetative heterokaryon they must carry matching alleles at a series of loci variously termed het or vic genes. Cloned het/vic genes from Neurospora crassa and Podospora anserina have no obvious functional similarity and have various cellular functions. Our objective was to identify the homologue of the Neurospora het-c gene in Fusarium proliferatum and to determine if this gene has a vegetative compatibility function in this economically important and widely dispersed fungal pathogen. In F. proliferatum and five other closely related Fusarium species we found a few differences in the DNA sequence, but the changes were silent and did not alter the amino acid sequence of the resulting protein. Deleting the gene altered sexual fertility as the female parent, but it did not alter male fertility or existing vegetative compatibility interactions. Replacement of the allele-specific portion of the coding sequence with the sequence of an alternate allele in N. crassa did not result in a vegetative incompatibility response in transformed strains of F. proliferatum. Thus, the fphch gene in Fusarium appears unlikely to have the vegetative compatibility function associated with its homologue in N. crassa. These results suggest that the vegetative compatibility phenotype may result from convergent evolution. Thus, the genes involved in this process may need to be identified at the species level or at the level of a group of species and could prove to be attractive targets for the development of antifungal agents.