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Mitochondrial genes in the colourless alga Prototheca wickerhamii resemble plant genes in their exons but fungal genes in their introns.

The mitochondrial DNA from the colourless alga Prototheca wickerhamii contains two mosaic genes as was revealed from complete sequencing of the circular extranuclear genome. The genes for the large subunit of the ribosomal RNA (LSUrRNA) as well as for subunit I of the cytochrome oxidase (coxI) carry two and three intronic sequences respectively. On the basis of their canonical nucleotide sequences they can be classified as group I introns. Phylogenetic comparisons of the coxI protein sequences allow us to conclude that the P.wickerhamii mtDNA is much closer related to higher plant mtDNAs than to those of the chlorophyte alga C.reinhardtii. The comparison of the intron sequences revealed several unusual features: (1) The P.wickerhamii introns are structurally related to mitochondrial introns from various ascomycetous fungi. (2) Phylogenetic analyses indicate a close relationship between fungal and algal intronic sequences. (3) The P. wickerhamii introns are located at positions within the structural genes which can be considered as preferred intron insertion sites in homologous mitochondrial genes from fungi or liverwort. In all cases, the sequences adjacent to the insertion sites are very well conserved over large evolutionary distances. Our finding of highly similar introns in fungi and algae is consistent with the idea that introns have already been present in the bacterial ancestors of present day mitochondria and evolved concomitantly with the organelles.

Amino Acid Sequence↗

Nutritional and growth control of ribosomal protein mRNA and rRNA in Neurospora crassa.

The effects of changing growth rates on the levels of 40S pre-rRNA and two r-protein mRNAs were examined to gain insight into the coordinate transcriptional regulation of ribosomal genes in the ascomycete fungus Neurospora crassa. Growth rates were varied either by altering carbon nutritional conditions, or by subjecting the isolates to inositol-limiting conditions. During carbon up- or down-shifts, r-protein mRNA levels were stoichiometrically coordinated. Changes in 40S pre-rRNA levels paralleled those of the r-protein mRNAs but in a non-stoichiometric manner. Comparison of crp-2 mRNA levels with those of a crp-2::qa-2 fusion gene indicated no major effect from changes in crp-2 mRNA stability. Crp-2 promoter mutagenesis experiments revealed that two elements of the crp-2 promoter, -95 to -83 bp (Dde box) and -74 to -66 bp (CG repeat) important for transcription under constant growth conditions, are also critical for transcriptional regulation by a carbon source. Ribosomal protein mRNA and rRNA levels were unaffected by changes in growth rates when the cultures were grown under inositol-limiting conditions, suggesting that, under these conditions, transcription of the ribosomal genes in N.crassa was regulated independently of growth rate.

Base Sequence↗

The Cryphonectria parasitica plasmid pUG1 contains a large ORF with motifs characteristic of family B DNA polymerases.

The isolation and characterization of the circular mitochondrial plasmid pUG1 from the ascomycete Cryphonectria parasitica is described. The entire sequence (4182 bp) was obtained and high similarities to DNA-dependent DNA polymerases were revealed. Strikingly common features with the DNA polymerases encoded by the Neurospora intermedia plasmids Fiji and LaBelle, such as matches to the conserved motifs A and B and the presence of TTD instead of DTD in motif C, were found, suggesting the existence of a distinct group of members of the B DNA family polymerases. These strong similarities between the plasmids might suggest a common origin of the C.parasitica and the Neurospora plasmids.

Amino Acid Sequence↗

Restriction endonuclease cleavage map of mitochondrial DNA from Aspergillus nidulans.

Mitochondrial DNA of the ascomycete fungus Aspergillus nidulans, a circular molecule of 31 500 base pairs, is cleaved by restriction endonucleases Eco R I, Hind II, Hind III and Bgl II into 3, 7, 9 and 5 fragments, respectively. The relative positions of the cleavage sites could be mapped by analysis of fragments obtained by double enzyme digestions of whole DNA and by complete and partial redigestion of isolated restriction fragments.

Aspergillus nidulans↗

Nucleotide sequence of 5S ribosomal RNA from Aspergillus nidulans and Neurospora crassa.

The nucleotide sequences of 5S rRNA molecules isolated from the cytosol and the mitochondria of the ascomycetes A. nidulans and N. crassa were determined by partial chemical cleavage of 3'-terminally labelled RNA. The sequence identity of the cytosolic and mitochondrial RNA preparations confirms the absence of mitochondrion-specific 5S rRNA in these fungi. The sequences of the two organisms differ in 35 positions, and each sequence differs from yeast 5S rRNA in 44 positions. Both molecules contain the sequence GCUC in place of GAAC or GAUY found in all other 5S rRNAs, indicating that this region is not universally involved in base-pairing to the invariant GTpsiC sequence of tRNAs.

Aspergillus nidulans↗

A comparison of three fission yeast mitochondrial genomes.

The fission yeasts are members of the fungal order Schizosaccharomycetales, a candidate deep-diverging group within Ascomycota. Although a great deal of molecular information is available from Schizosaccharomyces pombe, a model eukaryote, very little is available from other members of this group. In order to better characterize mitochondrial genome evolution in this fungal lineage, the mitochondrial DNA (mtDNA) of two additional fission yeasts, Schizosaccharomyces octosporus and Schizosaccharomyces japonicus var. japonicus, was sequenced. Whereas the mtDNA of S.pombe is only 19 431 bp, the mtDNA of S.octosporus is 44 227 bp, and that of S.japonicus var. japonicus is over 80 kb. The size variation of these mtDNAs is due largely to non-coding regions. The gene content in the latter two mtDNAs is almost identical to that of the completely sequenced S.pombe mtDNA, which encodes 25 tRNA species, the large and small mitochondrial ribosomal RNAs (rnl and rns), the RNA component of mitochondrial RNaseP (rnpB), mitochondrial small subunit ribosomal protein 3 (rps3), cytochrome oxidase subunits 1, 2 and 3 (cox1, cox2 and cox3) and ATP-synthase subunits 6, 8 and 9 (atp6, atp8 and atp9). However, trnI2(cau) (C modified to lysidine) is absent in the S.octosporus mtDNA, as are corresponding ATA codons in its protein-coding genes, and rps3 and rnpB are not found in the mtDNA of S.japonicus var. japonicus. The mtDNA of S.octosporus contains five double hairpin elements, the first report of these elements in an ascomycete. This study provides further evidence in favor of the mobility of these elements, and supports their role in mitochondrial genome rearrangement. The results of our phylogenetic analysis support the monophyly of the Schizosaccharomycetales, but question their grouping within the Archiascomycota.

Base Sequence↗

Long-oligomer microarray profiling in Neurospora crassa reveals the transcriptional program underlying biochemical and physiological events of conidial germination.

To test the inferences of spotted microarray technology against a biochemically well-studied process, we performed transcriptional profiling of conidial germination in the filamentous fungus, Neurospora crassa. We first constructed a 70 base oligomer microarray that assays 3366 predicted genes. To estimate the relative gene expression levels and changes in gene expression during conidial germination, we analyzed a circuit design of competitive hybridizations throughout a time course using a Bayesian analysis of gene expression level. Remarkable consistency of mRNA profiles with previously published northern data was observed. Genes were hierarchically clustered into groups with respect to their expression profiles over the time course of conidial germination. A functional classification database was employed to characterize the global picture of gene expression. Consensus motif searches identified a putative regulatory component associated with genes involved in ribosomal biogenesis. Our transcriptional profiling data correlate well with biochemical and physiological processes associated with conidial germination and will facilitate functional predictions of novel genes in N.crassa and other filamentous ascomycete species. Furthermore, our dataset on conidial germination allowed comparisons to transcriptional mechanisms associated with germination processes of diverse propagules, such as teliospores of the phytopathogenic fungus Ustilago maydis and spores of the social amoeba Dictyostelium discoideum.

Animals↗

The natural history of the WRKY-GCM1 zinc fingers and the relationship between transcription factors and transposons.

WRKY and GCM1 are metal chelating DNA-binding domains (DBD) which share a four stranded fold. Using sensitive sequence searches, we show that this WRKY-GCM1 fold is also shared by the FLYWCH Zn-finger domain and the DBDs of two classes of Mutator-like element (MULE) transposases. We present evidence that they share a stabilizing core, which suggests a possible origin from a BED finger-like intermediate that was in turn ultimately derived from a C2H2 Zn-finger domain. Through a systematic study of the phyletic pattern, we show that this WRKY-GCM1 superfamily is a widespread eukaryote-specific group of transcription factors (TFs). We identified several new members across diverse eukaryotic lineages, including potential TFs in animals, fungi and Entamoeba. By integrating sequence, structure, gene expression and transcriptional network data, we present evidence that at least two major global regulators belonging to this superfamily in Saccharomyces cerevisiae (Rcs1p and Aft2p) have evolved from transposons, and attained the status of transcription regulatory hubs in recent course of ascomycete yeast evolution. In plants, we show that the lineage-specific expansion of WRKY-GCM1 domain proteins acquired functional diversity mainly through expression divergence rather than by protein sequence divergence. We also use the WRKY-GCM1 superfamily as an example to illustrate the importance of transposons in the emergence of new TFs in different lineages.

Amino Acid Sequence↗

Evolutionary dynamics of multiple group I introns in nuclear ribosomal RNA genes of endoparasitic fungi of the genus Cordyceps.

A large number of group I introns were discovered in coding regions of small and large subunits of nuclear ribosomal RNA genes (SSU rDNA and LSU rDNA) in ascomycetous fungi of the genus CORDYCEPS: From 28 representatives of the genus, we identified in total 69 group I introns which were inserted at any of four specific sites in SSU rDNA and four specific sites in LSU rDNA. These group I introns reached sizes of up to 510 bp, occurred in up to eight sites in the same organism, and belonged to either subgroup IB3 or subgroup IC1 based on their sequence and structure. Introns inserted at the same site were closely related to each other among Cordyceps fungi, whereas introns inserted at different sites were phylogenetically distinct even in the same species. Mapped on the host phylogeny, the group I introns were generally not restricted to a particular lineage, but, rather, widely and sporadically distributed among distinct lineages. When the phylogenetic relationships of introns inserted at the same site were compared with the phylogeny of their hosts, the topologies were generally significantly congruent to each other. From these results, the evolutionary dynamics of multiple group I introns in Cordyceps fungi was inferred as follows: (1) most of the group I introns were already present at the eight sites in SSU and LSU rDNAs of the ancestor of the genus Cordyceps; (2) the introns have principally been immobile and vertically transmitted throughout speciation and diversification of Cordyceps fungi, which resulted in the phylogenetic congruence between the introns at the same site and their hosts; (3) in the course of vertical transmission, the introns have repeatedly been lost in a number of lineages independently, which has led to the present sporadic phylogenetic distribution of the introns; and (4) a few acquisitions of new introns, presumably through horizontal transmission, were identified in the evolutionary history of the genus Cordyceps, while no transpositions were detected. Losses of group I introns in SSU rDNA have occurred at least 27 times in the evolutionary course of the 28 Cordyceps members.

Base Sequence↗

Evolution of the Fot1 transposons in the genus Fusarium: discontinuous distribution and epigenetic inactivation.

To understand the evolution of Fot1, a member of the pogo family widely dispersed in ascomycetes, we have performed a phylogenetic survey across the genus Fusarium divided into six sections. The taxonomic distribution of Fot1 is not homogeneous but patchy; it is prevalent in the Fusarium oxysporum complex, absent in closely related sections, and found in five species from the most distant section Martiella. Multiple copies of Fot1 were sequenced from each strain in which the element occurs. In three species, the Fot1 nucleotide sequence is 98% identical to that from F. oxysporum (Fox), whereas nucleotide divergence for host genes is markedly higher: 11% for partial nuclear 28S rDNA and up to 30% for the gene encoding nitrate reductase (nia). In two species, sequence divergence of Fot1-related elements relative to Fox ranged from 7% to 23% (16% average). Most of the sequence differences (82%) were C-to-T and G-to-A transitions. These mutations are distributed throughout the Fot1 sequences, although they tend to be concentrated in the middle portion of the elements. Analysis of the local sequence context of transitions revealed a hierarchy of site preferences. These characteristics are typical of the repeat-induced point mutation process, first discovered in Neurospora crassa. The spotty distribution of Fot1 elements among species together with the high degree of similarity between Fot1 sequences present in distant species strongly suggests a case of horizontal transfer.

Base Sequence↗

RFLP markers show genetic recombination in Botryotinia fuckeliana (Botrytis cinerea) and transposable elements reveal two sympatric species.

Molecular markers revealed that Botryotinia fuckeliana (the teleomorph of Botrytis cinerea), a haploid, filamentous, heterothallic ascomycete, contained a large amount of intrapopulation genetic variation. The markers were used to determine the mode of reproduction and the population structure of this fungus. We did not detect any differentiation between isolates from different organs, collection dates, varieties of grape, or locations in the Champagne region of France, but two unexpected sympatric populations were identified. One group of isolates (transposa) contained the transposable elements Boty and Flipper; the other (vacuma) did not. These groups differed from one another for all the other markers. RFLP markers showed that there was genetic recombination in both groups of isolates. We conclude that there are two sympatric populations of B. fuckeliana in Champagne. One species (transposa) seems to be local and well adapted, while the other one (vacuma) is presumably a heterogeneous migrant population.

Alleles↗

Double-hairpin elements in the mitochondrial DNA of allomyces: evidence for mobility.

The mitochondrial DNA (mtDNA) of the chytridiomycete fungus Allomyces macrogynus contains 81 G+C-rich sequence elements that are 26-79 bases long and can be folded into a unique secondary structure consisting of two stem-loops. At the primary sequence level, the conservation of these double-hairpin elements (DHEs) is variable, ranging from marginal to complete identity. Forty of these DHEs are inserted in intergenic regions, 35 in introns, and 6 in variable regions of rRNA genes. Ten DHEs are inserted into other DHE elements (twins); two even form triplets. A comparison of DHE sequences shows that loop regions contain more sequence variation than helical regions and that the latter often contain compensatory base changes. This suggests a functional importance of the DHE secondary structure. We further identified nine DHEs in a 4-kb region of Allomyces arbusculus, a close relative of A. macrogynus. Eight of these DHEs are highly similar in sequence (90%-100%) to those in A. macrogynus, but only five are inserted at the same positions as in A. macrogynus. Interestingly, DHEs are also found in the mtDNAs of other chytridiomycetes, as well as certain zygomycete and ascomycete fungi. The overall distribution pattern of DHEs in fungal mtDNAs suggests that they are mobile elements.

Base Sequence↗

Interkingdom host jumping underground: phylogenetic analysis of entomoparasitic fungi of the genus cordyceps.

Most members of the ascomycetous genus Cordyceps are endoparasitic fungi of insects and other arthropods, but about 20 of the 300 described species are parasitic to hart's truffles, Elaphomyces spp. In order to understand the evolution of host specificity and the process of interkingdom host jumping in Cordyceps, we investigated the phylogenetic relationships of 22 representatives, including 4 truffle parasites and 18 insect parasites, based on nuclear and mitochondrial rDNA sequences. Five monophyletic groups were identified in both nuclear and mitochondrial phylogenies. In three of the five clades, the members utilized hosts from the same insect group, suggesting that the endoparasite-host connections have been conserved to some extent. On the other hand, it was also shown that major host shifts between distantly related insects must have occurred repeatedly. Notably, phylogenetic analyses strongly suggested that parasites of hart's truffles originated from parasites of cicada nymphs during the evolution of the CORDYCEPS: The common habitats of cicada nymphs and hart's truffles, deep underground and associated with tree roots, suggest that the interkingdom host jumping from Animalia to Fungi might have been promoted by the overlapping ecological niche of the unrelated hosts. This finding provides an impressive case of a drastic host shift in favor of the host habitat hypothesis.

Animals↗

Positions of multiple insertions in SSU rDNA of lichen-forming fungi.

Lichen-forming fungi, in symbiotic associations with algae, frequently have nuclear small subunit ribosomal DNA (SSU rDNA) longer than the 1,800 nucleotides typical for eukaryotes. The lichen-forming ascomycetous fungus Lecanora dispersa contains insertions at eight distinct positions of its SSU rDNA; the lichen-forming fungi Calicium tricolor and Porpidia crustulata each contain one insertion. Insertions are not limited to fungi that form lichens; the lichen ally Mycocalicium albonigrum also contains two insertions. Of the 11 insertion positions now reported for lichen-forming fungi and this ally, 6 positions are known only from lichen-forming fungi. Including the 4 newly reported in this study, insertions are now known from at least 17 positions among all reported SSU rDNA sequences. Insertions, most of which are Group I introns, are reported in fungal and protistan lineages and occur at corresponding positions in genomes as phylogenetically distant as the nuclei of fungi, green algae, and red algae. Many of these positions are exposed in the mature rRNA tertiary structure and may be subject to independent insertion of introns. Insertion of introns, accompanied by their sporadic loss, accounts for the scattered distribution of insertions observed within the SSU rDNA of these diverse organisms.

Base Sequence↗

Origin and evolution of organisms as deduced from 5S ribosomal RNA sequences.

A phylogenetic tree of most of the major groups of organisms has been constructed from the 352 5S ribosomal RNA sequences now available. The tree suggests that there are several major groups of eubacteria that diverged during the early stages of their evolution. Metabacteria (= archaebacteria) and eukaryotes separated after the emergence of eubacteria. Among eukaryotes, red algae emerged first; and, later, thraustochytrids (a Proctista group), ascomycetes (yeast), green plants (green algae and land plants), "yellow algae" (brown algae, diatoms, and chrysophyte algae), basidiomycetes (mushrooms and rusts), slime- and water molds, various protozoans, and animals emerged, approximately in that order. Three major types of photosynthetic eukaryotes--i.e., red algae (= Chlorophyll a group), green plants (Chl. a + b group) and yellow algae (Chl. a + c)--are remotely related to one another. Other photosynthetic unicellular protozoans--such as Cyanophora (Chl. a), Euglenophyta (Chl. a + b), Cryptophyta (Chl. a + c), and Dinophyta (Chl. a + c)--seem to have separated shortly after the emergence of the yellow algae.

Animals↗

MgHog1 regulates dimorphism and pathogenicity in the fungal wheat pathogen Mycosphaerella graminicola.

The dimorphic ascomycete pathogen Mycosphaerella graminicola switches from a yeastlike form to an infectious filamentous form that penetrates the host foliage through stomata. We examined the biological function of the mitogen-activated protein kinase-encoding gene MgHog1 in M. graminicola. Interestingly, MgHog1 mutants were unable to switch to filamentous growth on water agar that mimics the nutritionally poor conditions on the foliar surface and, hence, exclusively developed by a yeastlike budding process. Consequently, due to impaired initiation of infectious germ tubes, as revealed by detailed in planta cytological analyses, the MgHog1 mutants failed to infect wheat leaves. We, therefore, conclude that MgHog1 is a new pathogenicity factor involved in the regulation of dimorphism in M. graminicola. Furthermore, MgHog1 mutants are osmosensitive, resistant to phenylpyrrole and dicarboximide fungicides, and do not melanize.

Ascomycota↗

The a and b loci of Ustilago maydis hybridize with DNA sequences from other smut fungi.

The smut fungi are obligately parasitic during the sexual phase of their life cycle, and the mating-type genes of these fungi play key roles in both sexual development and pathogenicity. Among species of smut fungi it is common to find a bipolar mating system in which one locus with two alternate alleles is believed to control cell fusion and establishment of the infectious cell type. Alternatively, several species have a tetrapolar mating system in which two different genetic loci, one of which has multiple alleles, control fusion and subsequent development of the infection hyphae. Cloned sequences from the a and b mating-type loci of the tetrapolar smut fungus Ustilago maydis were used as hybridization probes to DNAs from 23 different fungal strains, including smut fungi with both tetrapolar and bipolar mating systems. In general, all of the smut fungi hybridized with the mating-type genes from U. maydis, suggesting conservation of the sequences involved in mating interactions. A selection of DNAs from other ascomycete and basidiomycete fungi failed to hybridize with the U. maydis mating-type sequences. Exceptions to this finding include hybridization of DNA from the a1 idiomorph of U. maydis to DNA from one strain of U. violacea and hybridization of both a idiomorphs to DNA from Saccharomyces cerevisiae.

Genes, Fungal↗

Analysis of determinants of binding and transcriptional activation of the pisatin-responsive DNA binding factor of Nectria haematococca.

Pisatin is a fungistatic isoflavonoid produced by garden pea. Field isolates of the ascomycete Nectria haematococca mating population VI (anamorph: Fusarium solani) that are highly virulent on pea have been found to possess the PDA1 gene encoding a pisatin detoxifying activity. Expression of PDA1 is specifically and highly induced by exposure of mycelia to pisatin. A pisatin-responsive DNA-binding activity has previously been identified with properties suggestive of a transcriptional regulator of PDA1. In this study, the sequence determinants for binding this pisatin-responsive factor (PRF) were localized to a 14-bp region through analysis of sequence alterations that reduced PRF binding. Using a homologous in vitro transcription system, a transcriptional activator of PDA1 was shown to be present in mycelial extracts that shared the sequence specificity characteristic of the PRF, indicating function of the DNA-binding protein in transcriptional control. A 70-kDa protein was shown to be a DNA-binding component of PRF by three independent assays for DNA-binding proteins: Southwestern (DNA-protein) blotting, UV-crosslinking, and binding to immobilized DNA. These results characterize a transcriptional activator acting on the PDA1 promoter that is responsive to a host-specific compound and provide insight into the regulation of fungal genes in response to plant flavonoids.

Base Sequence↗