Eric U. Selker

Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae

The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation.

Rapid SNP Discovery and Genetic Mapping Using Sequenced RAD Markers

Single nucleotide polymorphism (SNP) discovery and genotyping are essential to genetic mapping. There remains a need for a simple, inexpensive platform that allows high-density SNP discovery and genotyping in large populations. Here we describe the sequencing of restriction-site associated DNA (RAD) tags, which identified more than 13,000 SNPs, and mapped three traits in two model organisms, using less than half the capacity of one Illumina sequencing run. We demonstrated that different marker densities can be attained by choice of restriction enzyme. Furthermore, we developed a barcoding system for sample multiplexing and fine mapped the genetic basis of lateral plate armor loss in threespine stickleback by identifying recombinant breakpoints in F2 individuals. Barcoding also facilitated mapping of a second trait, a reduction of pelvic structure, by in silico re-sorting of individuals. To further demonstrate the ease of the RAD sequencing approach we identified polymorphic markers and mapped an induced mutation in Neurospora crassa. Sequencing of RAD markers is an integrated platform for SNP discovery and genotyping. This approach should be widely applicable to genetic mapping in a variety of organisms.

A histone H3 methyltransferase controls DNA methylation in Neurospora crassa

DNA methylation is involved in epigenetic processes such as X-chromosome inactivation, imprinting and silencing of transposons. We have demonstrated previously that dim-2 encodes a DNA methyltransferase that is responsible for all known cytosine methylation in Neurospora crassa. Here we report that another Neurospora gene, dim-5, is required for DNA methylation, as well as for normal growth and full fertility. We mapped dim-5 and identified it by transformation with a candidate gene. The mutant has a nonsense mutation in a SET domain of a gene related to histone methyltransferases that are involved in heterochromatin formation in other organisms. Transformation of a wild-type strain with a segment of dim-5 reactivated a silenced hph gene, apparently by ‘quelling’ of dim-5. We demonstrate that recombinant DIM-5 protein specifically methylates histone H3 and that replacement of lysine 9 in histone H3 with either a leucine or an arginine phenocopies the dim-5 mutation. We conclude that DNA methylation depends on histone methylation.

Lessons from the Genome Sequence of Neurospora crassa: Tracing the Path from Genomic Blueprint to Multicellular Organism

SUMMARY We present an analysis of over 1,100 of the ∼10,000 predicted proteins encoded by the genome sequence of the filamentous fungus Neurospora crassa. Seven major areas of Neurospora genomics and biology are covered. First, the basic features of the genome, including the automated assembly, gene calls, and global gene analyses are summarized. The second section covers components of the centromere and kinetochore complexes, chromatin assembly and modification, and transcription and translation initiation factors. The third area discusses genome defense mechanisms, including repeat induced point mutation, quelling and meiotic silencing, and DNA repair and recombination. In the fourth section, topics relevant to metabolism and transport include extracellular digestion; membrane transporters; aspects of carbon, sulfur, nitrogen, and lipid metabolism; the mitochondrion and energy metabolism; the proteasome; and protein glycosylation, secretion, and endocytosis. Environmental sensing is the focus of the fifth section with a treatment of two-component systems; GTP-binding proteins; mitogen-activated protein, p21-activated, and germinal center kinases; calcium signaling; protein phosphatases; photobiology; circadian rhythms; and heat shock and stress responses. The sixth area of analysis is growth and development; it encompasses cell wall synthesis, proteins important for hyphal polarity, cytoskeletal components, the cyclin/cyclin-dependent kinase machinery, macroconidiation, meiosis, and the sexual cycle. The seventh section covers topics relevant to animal and plant pathogenesis and human disease. The results demonstrate that a large proportion of Neurospora genes do not have homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease.

Transgene silencing of the al-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on DNA-DNA interactions or DNA methylation.

The molecular mechanisms involved in transgene‐induced gene silencing (‘quelling’) in Neurospora crassa were investigated using the carotenoid biosynthetic gene albino‐1 (al‐1) as a visual marker. Deletion derivatives of the al‐1 gene showed that a transgene must contain at least approximately 132 bp of sequences homologous to the transcribed region of the native gene in order to induce quelling. Transgenes containing only al‐1 promoter sequences do not cause quelling. Specific sequences are not required for gene silencing, as different regions of the al‐1 gene produced quelling. A mutant defective in cytosine methylation (dim‐2) exhibited normal frequencies and degrees of silencing, indicating that cytosine methylation is not responsible for quelling, despite the fact that methylation of transgene sequences frequently is correlated with silencing. Silencing was shown to be a dominant trait, operative in heterokaryotic strains containing a mixture of transgenic and non‐transgenic nuclei. This result indicates that a diffusable, trans‐acting molecule is involved in quelling. A transgene‐derived, sense RNA was detected in quelled strains and was found to be absent in their revertants. These data are consistent with a model in which an RNA‐DNA or RNA‐RNA interaction is involved in transgene‐induced gene silencing in Neurospora.

Rearrangement of duplicated DNA in specialized cells of Neurospora

Introduction of DNA into Neurospora crassa can lead to sequence instability in the sexual phase of the life cycle. Sequence instability was investigated by using a set of strains transformed with single copies of a plasmid including host sequences, Neurospora sequences deleted from the host genome, and foreign sequences. The sequences already represented in the host were rearranged at high frequency in a cross. In general, both elements of the duplication, that from the plasmid and that from the host, became rearranged, whether or not they were linked. Unique sequences were left unaltered. Cytosine residues in the rearranged sequences typically became methylated de novo. Results from tetrad analyses indicated that the rearrangements occur before meiosis, during a stage between fertilization and karyogamy. We suggest that this previously unrecognized genetic process, RIP (rearrangement induced premeiotically), may contribute diversity for evolution and also maintain the gross organization of the genome.

Use of a bacterial hygromycin B resistance gene as a dominant selectable marker in Neurospora crassa transformation

Dominant transformation markers allow maximum flexibility in the choice of transformation recipients. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. Authors C. Staben, B. Jensen, M. Singer, J. Pollock, M. Schechtman, J. Kinsey, and E. Selker This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol36/iss1/22 Staben, C.^1, B. Jensen^2, M. Singer^2 Dominant transformation markers allow maximum flexibility in the choice of transformation J. Pollock^3. M. Schechtman^3,4, recipients. The most widely used transformation marker in N. crassa has been the Neurospora gene J. Kinsey^5 and E. Selker^2 conferring benomyl resistance (Orbach, et al. 1986 Mol. Cell. Biol. 6:2456-2461). UnfortunateUse of a bacterial Hygromycin B resistance gene as a dominant ly, this marker is usually inactivated during sexual crosses, presumably because its introduction by transformation generates strains with duplicate ß-tubulin sequences that are subject to selectable marker in Neurospora inactivation during the sexual cycle by RIP (Selker et al. 1987 Cell 51:741-752; Selker and crassa transformation. Garrett, 1988 Proc. Natl. Acad. Sci. USA 85:68706874). We therefore looked for a dominant drug resistance marker that would not be homologous to any sequence in the Neurospora genome. A number of researchers have found that a bacterial gene encoding hygromycin B resistance (hph, hygromycin B phosphotransferase), if driven by a eukaryotic promoter, can confer drug resistance on eukaryotic cells. Hygromycin B (hygB) is an amino-glycoside that inhibits protein synthesis by causing mistranslation (Gonzalez et al. 1978 Biochim. Biophys. Acta 521:459-469) and by interfering with protein translocation (Singh et al. 1979 Nature 277:146-148). In fungi, Grits and Davies have had promising results in the use of the hph gene in yeast (Gritz and Davies 1983 Gene 29:179-188). Punt et al. and Cullen et al. have had success using this gene in both A. nidulans and A. niger (Punt et al. 1987 Gene 56:117-124; Cullen et al. 1987 Gene 57:21-26). Here we report the sensitivity of Neurospora to hygB and the use of two existing plasmids and derivatives as transformation markers in Neurospora. All of the strains of Neurospora that we have tested (approx. 15) are sensitive to hygB. We generally use Vogels minimal medium with sorbose for plates or sucrose for slants. We have selected resistance in a number of supplemented media, including a complete medium. HygB was obtained from Calbiochem. The commercial powder is dissolved in sterile water at 200 mg/ml and added to autoclaved media. Media can be stored for several months at 4°C. A number of different sensitivity tests have been performed. I n general, strains are sensitive to 150 ug/ml hygB. Several different plating conditions have been used to isolate transformants produced by standard procedures (Vollmer and Yanofsky 1986 Proc. Natl. Acad. Sci. USA 83:4869-4873). Our standard conditions involve plating 5 x 10^6 spheroplasts on a 25 ml sorbose plate (200 ug/ml hygB) in 8 ml top agar lacking hygB. Top and bottom layers usually contain 1.5% agar. Decreasing the volume of top agar or increasing the hygB concentration increases the stringency of the selection and improves the background, but discriminates against some transformants and lowers the apparent transformation frequency. We have used similar selection conditions to isolate hyg^r mutants from mutagenized conidia. The frequency of spontaneous mutation to hyg^r is not high enough to interfere with the selection. Our initial transformations used either pAN7-1 (Punt et al. 1987 Gene 56:117-124) or pDH25 (Cullen et al. 1987 Gene 57:21-26). These plasmids contain Aspergillus transcription signals that direct the expression of the E. coli hph gene. We have made derivatives of these plasmids that other researchers may find useful. Two of these, pCSN43 and pCSN44, contain a SalI fragment of pDH25 that confers resistance as well as pDH25. These plasmids provide additional unique cloning sites (Figure 1A, 1B). They offer the additional advantage that they replicate to a very high copy number in E. coli. The SalI fragment from pDH25 that is present in pCSN43 and pCSN44 has been transferred into several other plasmids, and has conferred hygB^r in each case. In pES200 the trpC terminator has been deleted from pDH25 leaving a single BamHI site (Figure 1C). This plasmid is as effective as pDH25 in conferring hygB resistance; the single BamHI site allows convenient cloning of BamHI compatible restriction fragments. We have not mapped the actual transcription signals or start sites that direct hph gene expression in N. crassa, though the structure-activity relationship for the trpC promoter has been studied in Aspergillus (Hamer and Timberlake 1987 Mol. Cell. Biol. 7:2352-2359). Surprisingly, insertion of a 430 bp TaqI fragment from the zeta region (Selker and Stevens, 1985 Proc. Natl. Acad. Sci. USA 82:8114-8118) into the ClaI site of pDH25, separating the promoter from the hph coding reion, did not abolish hygB^r. However, the ClaI-BamHI fragment containing the hph coding region is not sufficient for resistance. Plasmids pES200, pCSN43 and pCSN44 are available from the Fungal Genetics Stock Center.

Structural Basis for the Product Specificity of Histone Lysine Methyltransferases

DIM-5 is a SUV39-type histone H3 Lys9 methyltransferase that is essential for DNA methylation in N. crassa. We report the structure of a ternary complex including DIM-5, S-adenosyl-L-homocysteine, and a substrate H3 peptide. The histone tail inserts as a parallel strand between two DIM-5 strands, completing a hybrid sheet. Three post-SET cysteines coordinate a zinc atom together with Cys242 from the SET signature motif (NHXCXPN) near the active site. Consequently, a narrow channel is formed to accommodate the target Lys9 side chain. The sulfur atom of S-adenosyl-L-homocysteine, where the transferable methyl group is to be attached in S-adenosyl-L-methionine, lies at the opposite end of the channel, approximately 4 A away from the target Lys9 nitrogen. Structural comparison of the active sites of DIM-5, an H3 Lys9 trimethyltransferase, and SET7/9, an H3 Lys4 monomethyltransferase, allowed us to design substitutions in both enzymes that profoundly alter their product specificities without affecting their catalytic activities.

Diverse Pathways Generate MicroRNA-like RNAs and Dicer-Independent Small Interfering RNAs in Fungi

A variety of small RNAs, including the Dicer-dependent miRNAs and the Dicer-independent Piwi-interacting RNAs, associate with Argonaute family proteins to regulate gene expression in diverse cellular processes. These two species of small RNA have not been found in fungi. Here, by analyzing small RNAs associated with the Neurospora Argonaute protein QDE-2, we show that diverse pathways generate miRNA-like small RNAs (milRNAs) and Dicer-independent small interfering RNAs (disiRNAs) in this filamentous fungus. Surprisingly, milRNAs are produced by at least four different mechanisms that use a distinct combination of factors, including Dicers, QDE-2, the exonuclease QIP, and an RNase III domain-containing protein, MRPL3. In contrast, disiRNAs originate from loci producing overlapping sense and antisense transcripts, and do not require the known RNAi components for their production. Taken together, these results uncover several pathways for small RNA production in filamentous fungi, shedding light on the diversity and evolutionary origins of eukaryotic small RNAs.

The methylated component of the Neurospora crassa genome

Cytosine methylation is common, but not ubiquitous, in eukaryotes. Mammals and the fungus Neurospora crassa have about 2–3% of cytosines methylated. In mammals, methylation is almost exclusively in the under-represented CpG dinucleotides, and most CpGs are methylated whereas in Neurospora, methylation is not preferentially in CpG dinucleotides and the bulk of the genome is unmethylated. DNA methylation is essential in mammals but is dispensable in Neurospora, making this simple eukaryote a favoured organism in which to study methylation. Recent studies indicate that DNA methylation in Neurospora depends on one DNA methyltransferase, DIM-2 (ref. 6), directed by a histone H3 methyltransferase, DIM-5 (ref. 7), but little is known about its cellular and evolutionary functions. As only four methylated sequences have been reported previously in N. crassa, we used methyl-binding-domain agarose chromatography to isolate the methylated component of the genome. DNA sequence analysis shows that the methylated component of the genome consists almost exclusively of relics of transposons that were subject to repeat-induced point mutation—a genome defence system that mutates duplicated sequences.