Robust and efficient synthetic method for forming DNA microarrays.

The field of DNA microarray technology has necessitated the cooperative efforts of interdisciplinary scientific teams to achieve its primary goal of rapidly measuring global gene expression patterns. A collaborative effort was established to produce a chemically reactive surface on glass slide substrates to which unmodified DNA will covalently bind for improvement of cDNA microarray technology. Using the p-aminophenyl trimethoxysilane (ATMS)/diazotization chemistry that was developed, microarrays were fabricated and analyzed. This immobilization method produced uniform spots containing equivalent or greater amounts of DNA than commercially available immobilization techniques. In addition, hybridization analyses of microarrays made with ATMS/diazotization chemistry showed very sensitive detection of the target sequence, two to three orders of magnitude more sensitive than the commercial chemistries. Repeated stripping and re-hybridization of these slides showed that DNA loss was minimal, allowing multiple rounds of hybridization. Thus, the ATMS/diazotization chemistry facilitated covalent binding of unmodified DNA, and the reusable microarrays that were produced showed enhanced levels of hybridization and very low background fluorescence.

Meiotic silencing by unpaired DNA.

The silencing of gene expression by segments of DNA present in excess of the normal number is called cosuppression in plants and quelling in fungi. We describe a related process, meiotic silencing by unpaired DNA (MSUD). DNA unpaired in meiosis causes silencing of all DNA homologous to it, including genes that are themselves paired. A semidominant Neurospora mutant, Sad-1, fails to perform MSUD. Sad-1 suppresses the sexual phenotypes of many ascus-dominant mutants. MSUD may provide insights into the function of genes necessary for meiosis, including genes for which ablation in vegetative life would be lethal. It may also contribute to reproductive isolation of species within the genus Neurospora. The wild-type allele, sad-1(+), encodes a putative RNA-directed RNA polymerase.

The mating type locus of Neurospora crassa: identification of an adjacent gene and characterization of transcripts surrounding the idiomorphs.

Complexity in and around the A and a mating type idiomorphs in Neurospora crassa was examined. Six sets of transcripts surrounding the idiomorphs were identified by Northern analysis. Several different patterns of regulation were observed. The two pairs of transcripts closest to the centromere-proximal idiomorph flanks (variable regions) exhibited mating type-specific size differences. DNA sequence was obtained for the region surrounding the four transcripts which showed mating type-specific size and expression. One of these pairs encoded amino acid sequences highly similar to a domain present in the plasma membrane ATPase. A mutant allele of one of these genes was induced by repeat-induced point mutation (RIP), which resulted in altered perithecial development and the elimination of ascospore production. These transcripts comprise a cluster of genes which may be involved in the control of mating and sexual development in N. crassa.

A methylated Neurospora 5S rRNA pseudogene contains a transposable element inactivated by repeat-induced point mutation.

In an analysis of 22 of the roughly 100 dispersed 5S rRNA genes in Neurospora crassa, a methylated 5S rRNA pseudogene, Psi63, was identified. We characterized the Psi63 region to better understand the control and function of DNA methylation. The 120-bp 5S rRNA-like region of Psi63 is interrupted by a 1.9-kb insertion that has characteristics of sequences that have been modified by repeat-induced point mutation (RIP). We found sequences related to this insertion in wild-type strains of N. crassa and other Neurospora species. Most showed evidence of RIP; but one, isolated from the N. crassa host of Psi63, showed no evidence of RIP. A deletion from near the center of this sequence apparently rendered it incapable of participating in RIP with the related full-length copies. The Psi63 insertion and the related sequences have features of transposons and are related to the Fot1 class of fungal transposable elements. Apparently Psi63 was generated by insertion of a previously unrecognized Neurospora transposable element into a 5S rRNA gene, followed by RIP. We name the resulting inactivated Neurospora transposon PuntRIP1 and the related sequence showing no evidence of RIP, but harboring a deletion that presumably rendered it defective for transposition, dPunt.

Characterization of mat A-2, mat A-3 and deltamatA mating-type mutants of Neurospora crassa.

The mating-type locus of Neurospora crassa regulates mating identity and entry into the sexual cycle. The mat A idiomorph encodes three genes, mat A-1, mat A-2, and mat A-3. Mutations in mat A-1 result in strains that have lost mating identity and vegetative incompatibility with mat a strains. A strain containing mutations in both mat A-2 and mat A-3 is able to mate, but forms few ascospores. In this study, we describe the isolation and characterization of a mutant deleted for mat (deltamatA), as well as mutants in either mat A-2 or mat A-3. The deltamatA strain is morphologically wild type during vegetative growth, but it is sterile and heterokaryon compatible with both mat A and mat a strains. The mat A-2 and mat A-3 mutants are also normal during vegetative growth, mate as a mat A strain, and produce abundant biparental asci in crosses with mat a, and are thus indistinguishable from a wild-type mat A strain. These data and the fact that the mat A-2 mat A-3 double mutant makes few asci with ascospores indicate that MAT A-2 and MAT A-3 are redundant and may function in the same pathway. Analysis of the expression of two genes (sdv-1 and sdv-4) in the various mat mutants suggests that the mat A polypeptides function in concert to regulate the expression of some sexual development genes.

A putative rhamnogalacturonase required for sexual development of Neurospora crassa.

In previous work, the asd-I (ascus development) gene of the filamentous fingus Neurospora crassa was identified as a gene expressed preferentially during the sexual cycle and shown to be essential for normal sexual development. The asd-I gene has been sequenced and further characterized. It contains two introns, the first of which is in-frame and inefficiently or differentially spliced. The predicted ASD-I protein has extensive homology with rhamnogalacturonase B of Aspergillus aculeatus, which cleaves the backbone within the ramified hairy regions of pectin. In homozygous asd-I crosses, sexual development is initiated and large numbers of normal-sized asci are formed. Ascospore delineation does not occur, however, and no sexual progeny are produced. As most asd-I asci contain eight nuclei, the two meiotic divisions and subsequent mitotic division typical of normal crosses seem to occur, but the haploid nuclei are not partitioned into ascospores. In wild-type crosses, the ASD-I protein is present in large amounts in croziers and young asci, but it is only faintly detectable in more mature asci containing developing ascospores. Models to explain the possible role of a rhamnogalacturonase in sexual development are presented.

Loss of growth polarity and mislocalization of septa in a Neurospora mutant altered in the regulatory subunit of cAMP-dependent protein kinase.

In filamentous fungi, growth polarity (i.e. hyphal extension) and formation of septa require polarized deposition of new cell wall material. To explore this process, we analyzed a conditional Neurospora crassa mutant, mcb, which showed a complete loss of growth polarity when incubated at the restrictive temperature. Cloning and DNA sequence analysis of the mcb gene revealed that it encodes a regulatory subunit of cAMP-dependent protein kinase (PKA). Unexpectedly, the mcb mutant still formed septa when grown at the restrictive temperature, indicating that polarized deposition of wall material during septation is a process that is, at least in part, independent of polarized deposition during hyphal tip extension. However, septa formed in the mcb mutant growing at the restrictive temperature are mislocalized. Both polarized growth and septation are actin-dependent processes, and a concentration of actin patches is observed at growing hyphal tips and sites where septa are being formed. In the mcb mutant growing at the restrictive temperature, actin patches are uniformly distributed over the cell cortex; however, actin patches are still concentrated at sites of septation. Our results suggest that the PKA pathway regulates hyphal growth polarity, possibly through organizing actin patches at the cell cortex.

Intracellular phosphate--water oxygen exchange measured by mass spectrometry.

To study, in vivo, potential P(i)-water oxygen exchange catalyzed by each of two high-affinity P(i) symporters of Neurospora crassa, we have developed methods for the purification of P(i) from whole-cell extracts and the subsequent derivatization of P(i) for analysis by GC-MS. We have also modified a published procedure for the preparation of 18O-P(i). However, the high background rate of transport-independent oxygen exchange, determined by monitoring the appearance of 18O-P(i) in cells incubated in the presence of H(2)18O, masks detection of any transport-dependent oxygen exchange which may occur. The rate of intracellular P(i)-water oxygen exchange is 4.36 nmol 18O incorporated into P(i) per second per milligram of cell protein. The 18O isotopic distribution of the intracellular P(i) closely resembles that predicted for random exchange of a single P(i) oxygen with that of water per enzymatic event. Based upon the observed isotopic enrichment, we calculate that the bulk intracellular P(i) pool must undergo an average of one oxygen exchange per phosphate ion about every 3 s.

NUC-2, a component of the phosphate-regulated signal transduction pathway in Neurospora crassa, is an ankyrin repeat protein.

In response to phosphorus limitation, the fungus Neurospora crassa synthesizes a number of enzymes that function to bring more phosphate into the cell. The NUC-2 protein appears to sense the availability of phosphate and transmits the signal downstream to the regulatory pathway. The nuc-2+ gene has been cloned by its ability to restore growth of a nuc-2 mutant under restrictive conditions of high pH and low phosphate concentration. We mapped the cloned gene to the right arm of linkage group II, consistent with the chromosomal position of the nuc-2 mutation as determined by classical genetic mapping. The nuc-2' open reading frame is interrupted by five introns and codes for a protein of 1066 amino acid residues. Its predicted amino acid sequence has high similarity to that of its homolog in Saccharomyces cerevisiae, PHO81. Both proteins contain six ankyrin repeats, which have been implicated in the cyclin-dependent kinase inhibitory activity of PHO81. The phenotypes of a nuc-2 mutant generated by repeat-induced point mutation and of a strain harboring a UV-induced nuc-2 allele are indistinguishable. Both are unable to grow under the restrictive conditions, a phenotype which is to some degree temperature dependent. The nuc-2+ gene is transcriptionally regulated. A 15-fold increase in the level of the nuc-2+ transcript occurs in response to a decrease in exogenous phosphate concentration.

Escape from het-6 incompatibility in Neurospora crassa partial diploids involves preferential deletion within the ectopic segment.

Self-incompatible het-6OR/het-6PA partial diploids of Neurospora crassa were selected from a cross involving the translocation strain, T(IIL-->IIIR)AR18, and a normal sequence strain. About 25% of the partial diploids exhibited a marked increase in growth rate after 2 weeks, indicating that "escape" from het-6 incompatibility had occurred. Near isogenic tester strains with different alleles (het-6OR and het-6PA) were constructed and used to determine that 80 of 96 escape strains tested were het-6PA, retaining the het-6 allele found in the normal-sequence LGII position; 16 were het-6OR, retaining the allele in the translocated position. Restriction fragment length polymorphisms in 45 escape strains were examined with probes made from cosmids that spanned the translocated region. Along with electrophoretic analysis of chromosomes from three escape strains, RFLPs showed that escape is associated with deletion of part of one or the other of the duplicated DNA segments. Deletions ranged in size from approximately 70 kbp up to putatively the entire 270-kbp translocated region but always included a 35-kbp region wherein we hypothesize het-6 is located. The deletion spectrum at het-6 thus resembles other cases where mitotic deletions occur such as of tumor suppressor genes and of the hprt gene (coding for hypoxanthine-guanine phosphoribosyl-transferase) in humans.

Translocation of Neurospora crassa transcription factor NUC-1 into the nucleus is induced by phosphorus limitation.

NUC-1, a basic helix-loop-helix zipper protein, activates the expression of several genes involved in phosphorus acquisition in Neurospora crassa. In the present study we investigated whether posttranscriptional mechanisms control the activity of NUC-1. The NUC-1 level was higher (up to fivefold) in wild-type cells grown at low external phosphate concentration and in mutant strains expressing the phosphorus acquisition genes constitutively than in a wild-type strain grown at high external phosphate concentration. Using indirect immunofluorescence we demonstrated that NUC-1 is localized at least predominantly in the cytosol when wild-type N. crassa is grown with an adequate supply of phosphate, whereas NUC-1 is largely concentrated in the nucleus upon limitation of external phosphate. In mutant strains expressing the phosphorus acquisition genes constitutively, NUC-1 localization was also primarily in the nucleus. Thus, subcellular compartmentation of regulatory proteins is an important mechanism in regulating gene expression in filamentous fungi.

Meiotic transvection in fungi.

The Neurospora crassa Asm-1+ (ascospore maturation 1) gene encodes an abundant nucleus-localized protein required for formation of female structures and for ascospore maturation. Deletion mutants of Asm-1+ are "ascus-dominant," i.e., when crossed to wild type, neither Asm-1+ nor Asm-1 delta spores mature. To explain this behavior, we considered three models: an effect of reduced dosage of the gene product, failure of internuclear communication, and failure of transvection (regulation dependent on pairing of alleles). We found that for proper regulation of subsequent sexual sporulation, Asm-1+ must be in proximity, probably paired, to its allelic counterpart in the zygote: i.e., transvection must occur. Disruption of pairing causes failure of ascospore progeny to mature. Transvection in Neurospora, unlike in Drosophila, occurs immediately before meiosis, and can be demonstrated between wild-type alleles.

Activator-independent gene expression in Neurospora crassa.

A transgenic position effect that causes activator-independent gene expression has been described previously for three Neurospora crassa phosphate-repressible genes. We report analogous findings for two additional positively regulated genes, qa-2+ and ars-1+, indicating that such position effects are not limited to genes involved in phosphorus metabolism. In addition, we have characterized a number of mutants that display activator-independent gene expression. Each of these mutants contains a chromosomal rearrangement with one breakpoint located in the 5'-upstream region of the affected gene. This suggests that the rearrangements are associated with activator-independent gene expression and that these cis-acting mutations may represent a position effect similar to that responsible for rendering some transgenes independent of their transcriptional activators. We suggest that positively regulated genes in N. crassa are normally held in a transcriptionally repressed state by a cis-acting mechanism until specifically activated. Disruption of this cis-acting mechanism, either by random integration of a gene by transformation or by chromosomal rearrangement, renders these genes independent or partly independent of the transcriptional activator on which they normally depend.

Species-specific and mating type-specific DNA regions adjacent to mating type idiomorphs in the genus Neurospora.

Mating type idiomorphs control mating and subsequent sexual development in Neurospora crassa and were previously shown to be well conserved in other Neurospora species. The centromere-proximal flanks of the A and a idiomorphs, but not the distal flanks from representative heterothallic, pseudohomothallic, and homothallic Neurospora species contain apparent species-specific and/or mating type-specific sequences adjacent to the well-conserved idiomorphs. The variable flank is bordered by regions that are highly homologous in all species. The sequence of approximately 1 kb immediately flanking the conserved idiomorphs of each species was determined. Sequence identity between species ranged from 20% (essentially unrelated) to > 90%. By contrast, the mt-A1 gene shows 88-98% identity. Sequence and hybridization data also show that the centromere-proximal flanks are very different between the two mating types for N. intermedia, N. discreta, and N. tetrasperma, but not for N. sitophila and N. crassa. The data suggest a close evolutionary relationship between several of the species; this is suppported by phylogenetic analysis of their respective mt-A1 genes. The origin of the variable regions adjacent to the evolutionarily conserved mating type idiomorphs is unknown.

Sequence of the met-10+ locus of Neurospora crassa: homology to a sequence of unknown function in Saccharomyces cerevisiae chromosome 8.

We have determined the sequence of the Neurospora crassa met-10+ gene and its flanking regions, and have isolated and analyzed cDNA clones for this region. We have identified two closely linked genes transcribed in the same orientation. The met-10+ gene is the downstream gene; an open reading frame (ORF) derived from five exons encodes a 475-amino-acid protein. The deduced protein lacks similarity to other characterized proteins. However, it exhibits a strong similarity to the product of an ORF of unknown function on Saccharomyces cerevisiae chromosome 8. This sequence similarity suggests functional equivalence and should facilitate identification of the function of met-10+ using gene disruptions in S. cerevisiae.

Some property of the nucleus determines the competence of Neurospora crassa for transformation.

In Neurospora, transformation of spheroplasts is quite efficient and usually occurs with the transforming DNA integrated at ectopic sites in the chromosome. However, only a small fraction of the spheroplasts is actually competent for transformation. To distinguish whether the limitation to competence is at the level of the plasma membrane or at the level of the nucleus, we performed experiments in which heterocaryotic spheroplasts were required to integrate two different plasmids in one transformation procedure. The cotransformants were then analyzed to determine into which nucleus or nuclei the separate plasmids had integrated. Results of such experiments confirm that successful ectopic transformation in Neurospora crassa requires a competent nucleus. The integration patterns of the two separate plasmids indicate that the availability of appropriate chromosomal sites for ectopic integration may be an aspect of nuclear competence.

Repressible cation-phosphate symporters in Neurospora crassa.

The filamentous fungus Neurospora crassa possesses two nonhomologous high-affinity phosphate permeases, PHO-4 and PHO-5. We have isolated separate null mutants of these permeases, allowing us to study the remaining active transporter in vivo in terms of phosphate uptake and sensitivity to inhibitors. The specificity for the cotransported cation differs for PHO-4 and PHO-5, suggesting that these permeases employ different mechanisms for phosphate translocation. Phosphate uptake by PHO-4 is stimulated 85-fold by the addition of Na+, which supports the idea that PHO-4 is a Na(+)-phosphate symporter. PHO-5 is unaffected by Na+ concentration but is much more sensitive to elevated pH than is PHO-4. Presumably, PHO-5 is a H(+)-phosphate symporter. Na(+)-coupled symport is usually associated with animal cells. The finding of such a system in a filamentous fungus is in harmony with the idea that the fungal and animal kingdoms are more closely related to each other than either is to the plant kingdom.

Analysis of the DNA-binding and dimerization activities of Neurospora crassa transcription factor NUC-1.

NUC-1, a positive regulatory protein of Neurospora crassa, controls the expression of several unlinked target genes involved in phosphorus acquisition. The carboxy-terminal end of the NUC-1 protein has sequence similarity to the helix-loop-helix family of transcription factors. Bacterially expressed and in vitro-synthesized proteins, which consist of the carboxy-terminal portion of NUC-1, bind specifically to upstream sequences of two of its target genes, pho2+ and pho-4+. These upstream sequences contain the core sequence, CACGTG, a target for many helix-loop-helix proteins. A large loop region (47 amino acids) separates the helix I and helix II domains. Mutations and deletion within the loop region did not interfere with the in vitro or in vivo functions of the protein. Immediately carboxy-proximal to the helix II domain, the NUC-1 protein contains an atypical zipper domain which is essential for function. This domain consists of a heptad repeat of alanine and methionine rather than leucine residues. Analysis of mutant NUC-1 proteins suggests that the helix II and the zipper domains are essential for the protein dimerization, whereas the basic and the helix I domains are involved in DNA binding. The helix I domain, even though likely to participate in dimer formation while NUC-1 is bound to DNA, is not essential for in vitro dimerization.

Cloning and characterization of the pho-2+ gene encoding a repressible alkaline phosphatase in Neurospora crassa.

The Neurospora crassa phosphate-repressible alkaline phosphatase-encoding gene pho-2+ was cloned and its nucleotide sequence was determined. An open reading frame was found that contains four introns and encodes a putative protein of 555 amino acids. 'Activator-independent expression' of ectopically integrated pho-2+ was observed, as noted before for ectopically integrated pho-4+.