Rodolfo Aramayo

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.

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.

Meiotic silencing and the epigenetics of sex.

The sensing of accurate homologous recognition and pairing between discreet chromosomal regions and/or entire chromosomes entering meiosis is an essential step in ensuring correct alignment for recombination. A component of this is the recognition of heterology, which is required to prevent recombination at ectopic sites and between non-homologous chromosomes. It has been observed that a number of diverged organisms add an additional layer to this process: regions or chromosomes without a homologous counterpart are targeted for silencing during meiotic prophase I. This phenomenon was originally described in filamentous fungi, but has since been observed in nematodes and mammals. In this review we will generally group these phenomena under the title of meiotic silencing, and describe what is known about the process in the organisms in which it is observed. We will additionally propose that the functions of meiotic silencing originate in genome defense, and discuss its potential contributions to genome evolution and speciation.

The Aspergillus nidulans yA gene is regulated by abaA.

The developmentally regulated Aspergillus nidulans yA gene encodes a p‐diphenol oxidase that is needed for synthesis of green conidial pigment. We subjected the yA 5′ flanking region to mutational analysis in A. nidulans and Saccharomyces cerevisiae to identify DNA sequence elements involved in its transcriptional control, and identified two functionally distinct elements. Element I contained potential BrlA binding sites and was required for full level yA transcription, but not for developmental regulation in the presence of element II. Element II contained putative TEF‐1 binding sites flanking a CCAAT element and was sufficient for developmental regulation of transcription. Mutation of the TEF‐1 binding sites eliminated developmental regulation, whereas mutation of the CCAAT element led to elevated levels of transcription. Element II was also sufficient to induce transcription in S. cerevisiae when the A.nidulans developmental regulatory gene abaA was expressed from the GAL1 promoter. As AbaA and TEF‐1 possess similar DNA binding domains, the abaA‐yA interaction in yeast is probably direct. Thus, abaA appears to be a direct activator of yA, but yA regulation may also involve interactions with BrlA and a member of the CCAAT class of DNA binding proteins.

Properties of Unpaired DNA Required For Efficient Silencing in Neurospora crassa

The presence of unpaired copies of a gene during meiosis triggers silencing of all copies of the gene in the diploid ascus cell of Neurospora. This phenomenon is called meiotic silencing and on the basis of genetic studies appears to be a post-transcriptional gene silencing (PTGS) mechanism. Previously, meiotic silencing was defined to be induced by the presence of a DNA region lacking an identical segment in the homologous chromosome. However, the determinants of unpaired DNA remained a mystery. Using the Ascospore maturation-1 (Asm-1) gene, we defined what needs to be “unpaired” to silence a gene. For efficient silencing, an unpaired region of DNA needs to be of a sufficient size and contain homology to the reporter transcript. The greater the size of the loop and the larger the homology to the reporter transcript, the better the resulting meiotic silencing is. Conversely, regions not containing homology to the transcript, e.g., intergenic regions, did not silence the reporter. Surprisingly, unpaired fragments lacking a canonical promoter silenced the reporter. Additionally, we detected the unpairing-dependent loss of a transcript during meiotic silencing. Our observations further support a PTGS mechanism for meiotic silencing and offer insight into the evolutionary consequences resulting from this novel meiotic checkpoint.

A cytosine methyltransferase homologue is essential for sexual development in Aspergillus nidulans.

Background The genome defense processes RIP (repeat-induced point mutation) in the filamentous fungus Neurospora crassa, and MIP (methylation induced premeiotically) in the fungus Ascobolus immersus depend on proteins with DNA methyltransferase (DMT) domains. Nevertheless, these proteins, RID and Masc1, respectively, have not been demonstrated to have DMT activity. We discovered a close homologue in Aspergillus nidulans, a fungus thought to have no methylation and no genome defense system comparable to RIP or MIP. Principal Findings We report the cloning and characterization of the DNA methyltransferase homologue A (dmtA) gene from Aspergillus nidulans. We found that the dmtA locus encodes both a sense (dmtA) and an anti-sense transcript (tmdA). Both transcripts are expressed in vegetative, conidial and sexual tissues. We determined that dmtA, but not tmdA, is required for early sexual development and formation of viable ascospores. We also tested if DNA methylation accumulated in any of the dmtA/tmdA mutants we constructed, and found that in both asexual and sexual tissues, these mutants, just like wild-type strains, appear devoid of DNA methylation. Conclusions/Significance Our results demonstrate that a DMT homologue closely related to proteins implicated in RIP and MIP has an essential developmental function in a fungus that appears to lack both DNA methylation and RIP or MIP. It remains formally possible that DmtA is a bona fide DMT, responsible for trace, undetected DNA methylation that is restricted to a few cells or transient but our work supports the idea that the DMT domain present in the RID/Masc1/DmtA family has a previously undescribed function.

Neurospora crassa, a Model System for Epigenetics Research

The filamentous fungus Neurospora crassa has provided a rich source of knowledge on epigenetic phenomena that would have been difficult or impossible to gain from other systems. Neurospora sports features found in higher eukaryotes but absent in both budding and fission yeast, including DNA methylation and H3K27 methylation, and also has distinct RNA interference (RNAi)-based silencing mechanisms operating in mitotic and meiotic cells. This has provided an unexpected wealth of information on gene silencing systems. One silencing mechanism, named repeat-induced point mutation (RIP), has both epigenetic and genetic aspects and provided the first example of a homology-based genome defense system. A second silencing mechanism, named quelling, is an RNAi-based mechanism that results in silencing of transgenes and their native homologs. A third, named meiotic silencing, is also RNAi-based but is distinct from quelling in its time of action, targets, and apparent purpose.

Evidence for a precursor molecule of Brazil nut 2 S seed proteins from biosynthesis and cDNA analysis

SummarySeed storage proteins were extracted from Brazil nut (Bertholletia excelsa H.B.K.) seed embryos at various maturation stages. Salt-soluble and water-soluble proteins (globulins and albumins) were separated by gel chromatography and exhaustive dialysis against water. Both fractions were analysed by one- and two-dimensional polyacrylamide gel electrophoresis. Amino acid analysis revealed that both fractions are unusually high in methionine. The albumins consist of a family of low molecular weight polypeptides that are heterogeneous with respect to pI and are identical to the high methionine 2 S proteins described by Youle and Huang (1981). The biosynthesis of this class of proteins in maturing embryos was followed by in vivo labelling combined with immunological studies. Western blotting with monospecific antibodies against purified 2 S albumins and sequencing of a nearly complete cDNA clone revealed that they are synthesized via a precursor polypeptide.

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

In response to phosphorus limitation, the fungusNeurospora 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 down-stream to the regulatory pathway. Thenuc-2+ gene has been cloned by its ability to restore growth of anuc-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 thenuc-2 mutation as determined by classical genetic mapping. Thenuc-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 inSaccharomyces cerevisiae, PHO81. Both proteins contain six ankyrin repeats, which have been implicated in the cyclin-dependent kinase inhibitory activity of PHO81. The phenotypes of anuc-2 mutant generated by repeat-induced point mutation and of a strain harboring a UV-inducednuc-2 allele are indistinguishable. Both are unable to grow under the restrictive conditions, a phenotype which is to some degree temperature dependent. Thenuc-2+ gene is transcriptionally regulated. A 15-fold increase in the level of thenuc-2+ transcript occurs in response to a decrease in exogenous phosphate concentration.

The emerging world of wikis

We noted with interest the letter “Preserving accuracy in Gen Bank,” (M. I. Bidartondo et al. , 21 March, p. [1616][1]) and the related News of the Week story “Proposal to ‘wikify’ GenBank meets stiff resistance” (E. Pennisi, 21 March, p. [1598][2]). David Lipmans fears that wikifying