Many Basidiomycete genomes include substantial fractions that are deficient in CG dinucleotides, in extreme cases amounting to 70% of the genome. CG deficiency is variable and correlates with genome size and, more closely, with transposable element (TE) content. Many species have limited CG deficiency; it is therefore likely that there are other mechanisms that can control TE proliferation. Examination of TEs confirms that C-to-T transition mutations in CG dinucleotides may comprise a conspicuous proportion of differences between paired elements, however transition/transversion ratios are never as high as those due to RIP in some Ascomycetes, suggesting that repeat-associated CG mutation is not totally pervasive. This has allowed gene family expansion in Basidiomycetes, although CG transition differences are often prominent in paired gene family members, and are evidently responsible for destruction of some copies. A few lower fungal genomes exhibit similar evidence of repeat-associated CG mutation, as do the genomes of the two lower plants Physcomitrella patens and Selaginella moellendorffii, in both of which mutation parallels published methylation of CHG as well as CG nucleotides. In Basidiomycete DNA methylation has been reported to be largely confined to CG dinucleotides in repetitive DNA, but while methylation and mutation are evidently associated, it is not clear which is cause and which effect.

Thermostable enzymes and thermophilic cell factories may afford economic advantages in the production of many chemicals and biomass-based fuels. Here we describe and compare the genomes of two thermophilic fungi, Myceliophthora thermophila and Thielavia terrestris. To our knowledge, these genomes are the first described for thermophilic eukaryotes and the first complete telomere-to-telomere genomes for filamentous fungi. Genome analyses and experimental data suggest that both thermophiles are capable of hydrolyzing all major polysaccharides found in biomass. Examination of transcriptome data and secreted proteins suggests that the two fungi use shared approaches in the hydrolysis of cellulose and xylan but distinct mechanisms in pectin degradation. Characterization of the biomass-hydrolyzing activity of recombinant enzymes suggests that these organisms are highly efficient in biomass decomposition at both moderate and high temperatures. Furthermore, we present evidence suggesting that aside from representing a potential reservoir of thermostable enzymes, thermophilic fungi are amenable to manipulation using classical and molecular genetics.

The genomes of 49 filamentous ascomycetes (subphylum Pezizomycotina) were examined by two independent methods for evidence of multiple C→T transitions typical of RIP. At least one transposable element or other repeat family was identified in each genome, and members were assessed for transition and transversion mutations relative to a model of their intact progenitor. Occurrence of RIP was indicated where family members differed by excess of directional transitions over transversions. Transition mutations were quantified by an algorithm taking double mutations in CpG and CpC dinucleotides into account. A second method assessed dinucleotide frequency distribution anomalies in whole genomes, a procedure that allowed quantification of fractions of the non-coding genome that had been subject to extensive directional mutation. The results of both methods revealed that RIP-like activity varied greatly, both in extent of mutation and in dinucleotide context for C→T transitions. In the most extreme case, 75% of a Blastomyces dermatitidis genome had suffered conspicuous GC-depletion, all of it in the non-coding fraction. Many genomes carried both intact repeats as well as others that had suffered heavily from transitions. Only one species, Chaetomium globosum, showed no evidence of directional mutation.

The identification and annotation of protein-coding genes is one of the primary goals of whole-genome sequencing projects, and the accuracy of predicting the primary protein products of gene expression is vital to the interpretation of the available data and the design of downstream functional applications. Nevertheless, the comprehensive annotation of eukaryotic genomes remains a considerable challenge. Many genomes submitted to public databases, including those of major model organisms, contain significant numbers of wrong and incomplete gene predictions. We present a community-based reannotation of the Aspergillus nidulans genome with the primary goal of increasing the number and quality of protein functional assignments through the careful review of experts in the field of fungal biology.

An article in this issue of Molecular Microbiology by Cultrone et al. describes how a non-autonomous helitron element could arise from its autonomous parent transposon by deletion followed by readthrough into an adjacent gene and its promoter, thus providing a mechanism for distribution of a specifically regulated promoter sequence around the genome, where it would have the potential to evolve new functions.

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.

The sequences of five MATE transposable elements were retrieved from the Aspergillus nidulans genome sequence. These elements are 6.1 kb in length and are characterized by 9-10 bp target site duplications, paired approximately 40 bp palindromes close to each end, and in the unmutated elements, 57 clustered Spe-motifs (RWCTAGWY) scattered through their length. Short open reading frames have no known homology. Two of the MATE elements have numerous C --> T transitions on both DNA strands relative to the remaining three elements. These mutations have all the characteristics of repeat-induced point mutation (RIP) previously described in Neurospora crassa, but not experimentally demonstrated in A. nidulans. Ninety-eight percent of mutated cytosines are in CpG and CpA doublets, the former mutating at higher frequency.

Parasexual recombination is a valuable tool in the laboratory, particularly for asexual fungi, and a number of developments in methodology are outlined. In biotechnology, the parasexual cycle has proved less useful than at one time predicted, but it retains a function in analysis of the products of genetic manipulation, and as a convenient detection system for environmental chemicals that may disturb mitosis. In nature, recent evidence suggests that parasexual recombination is rare, in part at least because of the prevalence of heterokaryon incompatibility of many wild fungi.

Anucleate primary sterigmata (aps) mutants of Aspergillus nidulans are partially blocked in conidiation (asexual sporulation) due to failure of the organized migration of nuclei into the conidiophore metulae. The mutants also have a slightly reduced hyphal growth rate and irregular distribution of nuclei in vegetative hyphae; the hyphal phenotype appears somewhat more variable than the conidiation defect. The mutants fall into two complementation groups, apsA and apsB, mapping on chromosomes IV and VI, respectively. apsB mutants are also partially defective in sexual reproduction.

We describe a new method of gene cloning by complementation of mutant alleles which obviates the need for construction of a gene library in a plasmid vector in vitro and its amplification in Escherichia coli. The method involves simultaneous transformation of mutant strains of the fungus Aspergillus nidulans with (i) fragmented chromosomal DNA from a donor species and (ii) DNA of a plasmid without a selectable marker gene, but with a fungal origin of DNA replication ('helper plasmid'). Transformant colonies appear as the result of the joining of chromosomal DNA fragments carrying the wild-type copies of the mutant allele with the helper plasmid. Joining may occur either by ligation (if the helper plasmid is in linear form) or recombination (if it is cccDNA). This event occurs with high efficiency in vivo, and generates an autonomously replicating plasmid cointegrate. Transformants containing Penicillium chrysogenum genomic DNA complementing A. nidulans niaD, nirA and argB mutations have been obtained. While some of these cointegrates were evidently rearranged or consisted only of unaltered replicating plasmid, in other cases plasmids could be recovered into E. coli and were subsequently shown to contain the selected gene. The utility of this ''instant gene bank'' technique is demonstrated here by the molecular cloning of the P. canescens trpC gene.

We have constructed an intragenic map for the Aspergillus nidulans brlA gene, mutants in which are distinguishable by visual criteria only. Most of the leaky phenotype mutants map near the right (3') end. The gene shows distinct recombinational polarity consistent with recombination initiation at the promoter (centromereproximal) end of the gene. BrlA12 and brlA20 mutants gave abnormal DNA restriction patterns consistent with the III; VIII and VI; VIII translocations, respectively, determined by haploidization.