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Genetic analysis of suppressors of the veA1 mutation in Aspergillus nidulans.

Light-dependent conidiation in the filamentous ascomycete, Aspergillus nidulans, is contingent on the allelic state of the velvet (veA) gene. Light dependence is abolished by a mutation in this gene (veA1), which allows conidiation to occur in the absence of light. We have isolated and characterized six extragenic suppressors of veA1 that restore the light-dependent conidiation phenotype. Alleles of four genes, defined by complementation tests, were subjected to extensive genetic and phenotypic analysis. The results of light-dark shifting experiments and the phenotypes of double mutant combinations are consistent with the possibility that the expression of the light-dependent phenotype is regulated by specific interactions of the suppressor gene products with the velvet gene product and with each other.

Alleles↗

A restriction fragment length polymorphism map and electrophoretic karyotype of the fungal maize pathogen Cochliobolus heterostrophus.

A restriction fragment length polymorphism (RFLP) map has been constructed of the nuclear genome of the plant pathogenic ascomycete Cochliobolus heterostrophus. The segregation of 128 RFLP and 4 phenotypic markers was analyzed among 91 random progeny of a single cross; linkages were detected among 126 of the markers. The intact chromosomal DNAs of the parents and certain progeny were separated using pulsed field gel electrophoresis and hybridized with probes used to detect the RFLPs. In this way, 125 markers were assigned to specific chromosomes and linkages among 120 of the markers were confirmed. These linkages totalled 941 centimorgans (cM). Several RFLPs and a reciprocal translocation were identified tightly linked to Tox1, a locus controlling host-specific virulence. Other differences in chromosome arrangement between the parents were also detected. Fourteen gaps of at least 40 cM were identified between linkage groups on the same chromosomes; the total map length was therefore estimated to be, at a minimum, 1501 cM. Fifteen A chromosomes ranging from about 1.3 megabases (Mb) to about 3.7 Mb were identified; one of the strains also has an apparent B chromosome. This chromosome appears to be completely dispensable; in some progeny, all of 15 markers that mapped to this chromosome were absent. The total genome size was estimated to be roughly 35 Mb. Based on these estimates of map length and physical genome size, the average kb/cM ratio in this cross was calculated to be approximately 23. This low ratio of physical length to map distance should make this RFLP map a useful tool for cloning genes.

Ascomycota↗

Evolutionary dynamics of spore killers.

Spore killing in ascomycetes is a special form of segregation distortion. When a strain with the Killer genotype is crossed to a Sensitive type, spore killing is expressed by asci with only half the number of ascospores as usual, all surviving ascospores being of the Killer type. Using population genetic modeling, this paper explores conditions for invasion of Spore killers and for polymorphism of Killers, Sensitives and Resistants (which neither kill, nor get killed), as found in natural populations. The models show that a population with only Killers and Sensitives can never be stable. The invasion of Killers and stable polymorphism only occur if Killers have some additional advantage during the process of spore killing. This may be due to the effects of local sib competition or some kind of "heterozygous" advantage in the stage of ascospore formation or in the short diploid stage of the life cycle. This form of segregation distortion appears to be essentially different from other, well-investigated forms, and more field data are needed for a better understanding of spore killing.

Biological Evolution↗

Altered mating-type identity in the fungus Podospora anserina leads to selfish nuclei, uniparental progeny, and haploid meiosis.

In wild-type crosses of the filamentous ascomycete Podospora anserina, after fertilization, only nuclei of opposite mating type can form dikaryons that undergo karyogamy and meiosis, producing biparental progeny. To determine the role played by the mating type in these steps, the four mat genes were mutagenized in vitro and introduced into a strain deleted for its mat locus. Genetic and cytological analyses of these mutant strain, crossed to each other and to wild type, showed that mating-type information is required for recognition of nuclear identity during the early steps of sexual reproduction. In crosses with strain carrying a mating-type mutation, two unusual developmental patterns were observed: monokaryotic cells, resulting in haploid meiosis, and uniparental dikaryotic cells providing, after karyogamy and meiosis, a uniparental progeny. Altered mating-type identity leads to selfish behavior of the mutant nucleus: it migrates alone or paired, ignoring its wild-type partner in all mutant x wild-type crosses. This behavior is nucleus-autonomous because, in the same cytoplasm, the wild-type nuclei form only biparental dikaryons. In P. anserina, mat genes are thus required to ensure a biparental dikaryotic state but appear dispensable for later stages, such as meiosis and sporulation.

Alleles↗

Isolation of the Ascobolus immersus spore color gene b2 and study in single cells of gene silencing by methylation induced premeiotically.

The ascomycete Ascobolus immersus has been extensively used as a model system for the genetic study of meiotic recombination. More recently, an epigenetic process, known as methylation induced premeiotically (MIP), that acts on duplicated sequences has been discovered in A. immersus and has raised a new interest in this fungus. To try and extend these studies, we have now cloned the A. immersus spore color gene b2, a well characterized recombination hot-spot. Isolation of the whole gene was verified by physical mapping of four large b2 alterations, followed by transformation and mutant rescue of a null b2 allele. Transformation was also used to duplicate b2 and subject it to MIP. As a result, we were able for the first time to observe gene silencing as early as just after meiosis and in single cells. Furthermore, we have found evidence for a modulating effect of MIP on b2 expression, depending on the region of the gene that is duplicated and hence subjected to MIP.

Ascomycota↗

Suppressed recombination and a pairing anomaly on the mating-type chromosome of Neurospora tetrasperma.

Neurospora crassa and related heterothallic ascomycetes produce eight homokaryotic self-sterile ascospores per ascus. In contrast, asci of N. tetrasperma contain four self-fertile ascospores each with nuclei of both mating types (matA and mata). The self-fertile ascospores of N. tetrasperma result from first-division segregation of mating type and nuclear spindle overlap at the second meiotic division and at a subsequent mitotic division. Recently, Merino et al. presented population-genetic evidence that crossing over is suppressed on the mating-type chromosome of N. tetrasperma, thereby preventing second-division segregation of mating type and the formation of self-sterile ascospores. The present study experimentally confirmed suppressed crossing over for a large segment of the mating-type chromosome by examining segregation of markers in crosses of wild strains. Surprisingly, our study also revealed a region on the far left arm where recombination is obligatory. In cytological studies, we demonstrated that suppressed recombination correlates with an extensive unpaired region at pachytene. Taken together, these results suggest an unpaired region adjacent to one or more paired regions, analogous to the nonpairing and pseudoautosomal regions of animal sex chromosomes. The observed pairing and obligate crossover likely reflect mechanisms to ensure chromosome disjunction.

Base Sequence↗

Spore-killing meiotic drive factors in a natural population of the fungus Podospora anserina.

In fungi, meiotic drive is observed as spore killing. In the secondarily homothallic ascomycete Podospora anserina it is characterized by the abortion of two of the four spores in the ascus. We have identified seven different types of meiotic drive elements (Spore killers). Among 99 isolates from nature, six of these meiotic drive elements occurred in a local population. Spore killers comprise 23% of the natural population of P. anserina in Wageningen, The Netherlands, sampled from 1991 to 1997. One Spore-killer type was also found in a French strain dating from 1937. All other isolates found so far are sensitive to spore killing. All seven Spore killer types differ in the percentage of asci that show killing and in their mutual interactions. Interactions among Spore killer types showed either mutual resistance or dominant epistasis. Most killer elements could be assigned to linkage group III but are not tightly linked to the centromere.

Centromere↗

Genetic control of extracellular protease synthesis in the yeast Yarrowia lipolytica.

Depending on the pH of the growth medium, the yeast Yarrowia lipolytica secretes an acidic protease or an alkaline protease, the synthesis of which is also controlled by carbon, nitrogen, and sulfur availability, as well as by the presence of extracellular proteins. Previous results have indicated that the alkaline protease response to pH was dependent on YlRim101p, YlRim8p/YlPalF, and YlRim21p/YlPalH, three components of a conserved pH signaling pathway initially described in Aspergillus nidulans. To identify other partners of this response pathway, as well as pH-independent regulators of proteases, we searched for mutants that affect the expression of either or both acidic and alkaline proteases, using a YlmTn1-transposed genomic library. Four mutations affected only alkaline protease expression and identified the homolog of Saccharomyces cerevisiae SIN3. Eighty-nine mutations affected the expression of both proteases and identified 10 genes. Five of them define a conserved Rim pathway, which acts, as in other ascomycetes, by activating alkaline genes and repressing acidic genes at alkaline pH. Our results further suggest that in Y. lipolytica this pathway is active at acidic pH and is required for the expression of the acidic AXP1 gene. The five other genes are homologous to S. cerevisiae OPT1, SSY5, VPS28, NUP85, and MED4. YlOPT1 and YlSSY5 are not involved in pH sensing but define at least a second protease regulatory pathway.

Amino Acid Sequence↗

No detection of characteristic fungal protein elongation factor EF-3 in Pneumocystis carinii.

The taxonomic status of Pneumocystis carinii is uncertain, and P. carinii has been categorized both as a fungus and as a protozoan. Recent comparisons of RNA sequence homologies between P. carinii and several genera of fungi and protozoa suggest that P. carinii has closer affinities with the ascomycetes than with the protozoa. The translatory systems of the fungi, however, require three soluble protein factors for peptide chain elongation rather than the two necessary in other eukaryotic systems; to date the additional protein elongation factor (EF-3) appears to be unique to fungi. Western blot analysis of cell-free extracts of P. carinii, derived from rat, was done using a polyclonal antibody raised in rabbits to Saccharomyces cerevisiae EF-3. Anti-EF-3 cross-reacting material was detected only in lysates of Candida albicans and S. cerevisiae included as fungal controls; no cross reaction was detected in lysates of P. carinii, P. carinii-infected rat lung, or a protozoan control (Trichomonas vaginalis).

Animals↗

Elemental sulphur as an induced antifungal substance in plant defence.

Man's oldest fungicide has probably long functioned in this role in plants, as a natural component of induced antifungal defence. Elemental sulphur (S(0)) is the only inorganic phytoalexin and the only phytoalexin produced by so many different taxa. S(0) (detected by GC-MS as (32)S(8)) is produced in representative species of Sterculiaceae (cocoa), Solanaceae (tomato, tobacco), Malvaceae (cotton), and Leguminosae (French bean) in response to xylem-invading fungal and bacterial pathogens. Production was more rapid and intensive in disease-resistant genotypes. Gene expression for S(0) production may be xylem-specific as S(0) was not present in leaves of six species undergoing hypersensitivity to Pseudomonas syringae. Anomalously, high constitutive S(0) levels occurred in leaves of Arabidopsis and Brassica oleracea. S(0) was highly toxic (ED(50) 1-3 microg ml(-1)) to many fungal pathogens representing ascomycetes, basidiomycetes, and deuteromycetes, but not to an oomycete, Phytophthora, or to bacteria. Levels in cocoa and tomato xylem and Arabidopsis leaves were potentially inhibitory, but in other interactions were below theoretically toxic concentrations. However, S(0) accumulation is highly localized, suggesting that the element is produced in sufficient amounts, at the right time and place to be effective. SEM-EDX revealed S in tomato and cocoa xylem walls, xylem parenchyma, and vascular gels and tyloses, all sites appropriate to counter vascular pathogenic Verticillium dahliae. Transient increases in sulphate, glutathione and cysteine occurred in tomato xylem. The sulphate may reflect the over-expression of sulphate transporters, but the thiols might be possible precursors. Analysis of differential gene expression should reveal what may be a novel biosynthetic pathway of S(0) formation in eukaryotes.

Antifungal Agents↗

Hop, an active Mutator-like element in the genome of the fungus Fusarium oxysporum.

A new type of active DNA transposon has been identified in the genome of Fusarium oxysporum by its transposition into the niaD target gene. Two insertions within the final exon, in opposite orientations at the same nucleotide site, have been characterized. These elements, called Hop, are 3,299 bp long, with perfect terminal inverted repeats (TIRs) of 99 bp. The sequencing of genomic copies reveals a 9-bp target site duplication and no apparent sequence specificity at the insertion sites. The sequencing of a cDNA indicates that Hop does not contain an intron and encodes a putative transposase of 836 amino acids. The structural features (length, TIRs size, and 9-bp duplication), together with the presence of conserved domains in the transposase, strongly suggest that Hop is a Mutator-like element (MULE). Hop is thus the first active member of this family found beyond plants. The high rate of excision observed indicates that Hop is very active and thus represents a promising efficient tagging system for the isolation of fungal genes. The distribution of Hop elements within the Fusarium genus revealed that they are present in different species, suggesting that related elements could be present in other fungal genomes. In fact, Hop-related sequences have been identified in the survey of the entire genome sequence of three other ascomycetes, Magnaporthe grisea, Neurospora crassa, and Aspergillus fumigatus.

Amino Acid Sequence↗

Testing "species pair" hypotheses: evolutionary processes in the lichen-forming species complex Porpidia flavocoerulescens and Porpidia melinodes.

Pairs of taxa are commonly found in lichen-forming ascomycetes that differ primarily in their reproductive modes: one taxon reproduces sexually, the other vegetatively. The evolutionary processes underlying such "species pairs" are unknown. The species pair formed by Porpidia flavocoerulescens (sexual) and Porpidia melinodes (vegetative) was chosen to investigate four previously proposed hypotheses. These hypotheses posit that species pairs are either two monophyletic, independently evolving species with contrasting reproductive mode; a single outcrossing species polymorphic with regard to its reproductive modes; a sexual mother lineage frequently giving rise to asexual spin-offs; or a complex of cryptic species. The phylogenetic patterns observed within the species pair in the present study were analyzed using stringent hypothesis testing and visualizations of relationships and conflict based on tree and network reconstructions. DNA sequences at the three analyzed loci revealed the same four to five deeply divergent lineages. A detailed analysis of DNA-sequence variability revealed closely linked gene loci, but high levels of conflict within each of the gene fragments, as well as between observed genetic lineages. The observed patterns of phylogenetic relationships, linkage, and conflict are not congruent with any of the previously proposed species pair hypotheses. Rather, it is proposed that the observed results can be explained by conflicting reproductive and nutritional requirements imposed by an obligate symbiotic lifestyle. These interacting constraints produce recurring selective sweeps within predominantly vegetatively reproducing lineages and are the main forces that shape the evolution within the investigated species pair.

Base Sequence↗

Tallysomycin-induced mitotic aneuploidy and point mutations in Aspergillus nidulans.

Tallysomycin is an antibiotic compound structurally related to bleomycin, and like bleomycins and phleomycins also shows antitumour activity. We have investigated the genetic activity of tallysomycin in the ascomycete Aspergillus nidulans for malsegregation of chromosomes at mitosis and for point mutations. We found that the antibiotic at very low concentrations from 0.025 to 0.2 micrograms/ml had an inhibitory effect on colonial growth of up to 50% and it increased the number of mitotic malsegregants from 311% to 607% over the control value. Tallysomycin at concentrations between 2 and 16 micrograms/ml (which inhibited the germination of conidia from 30 to 90%) also increased the number of methionine suppressor revertants from 59 to 368 per 10(6) conidia. With regard to the mechanism for induced mitotic segregation it was shown that this may involve non-disjunction of chromosomes which through the intermediate formation of unstable aneuploids resulted in formation of stable haploid and diploid segregants.

Aneuploidy↗

Repeated DNA sequences in fungi.

Several fungal species, representatives of all broad groups like basidiomycetes, ascomycetes and phycomycetes, were examined for the nature of repeated DNA sequences by DNA:DNA reassociation studies using hydroxyapatite chromatography. All of the fungal species tested contained 10-20% repeated DNA sequences. There are approximately 100-110 copies of repeated DNA sequences of approximately 4 x 10(7) daltons piece size of each. Repeated DNA sequence homoduplexes showed on average 5 degrees C difference of T(e)50 (temperature at which 50% duplexes dissociate) values from the corresponding homoduplexes of unfractionated whole DNA. It is suggested that a part of repetitive sequences in fungi constitutes mitochondrial DNA and a part of it constitutes nuclear DNA.

DNA, Fungal↗

5S rRNA sequences from eight basidiomycetes and fungi imperfecti.

The 5S rRNA sequences from the basidiomycetes or fungi imperfecti Rhizoctonia crocorum, Rhizoctonia hiemalis, Exobasidium vaccinii, Trichosporon oryzae, Tilletia controversa, Tilletiaria anomala, Dacrymyces deliquescens and Coprinus radiatus were determined. With the exception of Exobasidium, these sequences conform to the association previously found between septal pore type and sequence. The sequence from the supposed ascomycete anamorph Rhizotonia hiemalis clearly is allied with basidiomycete sequences.

Base Sequence↗

Correct removal by splicing of a Neurospora intron in yeast.

Processing of intron-containing nuclear messenger RNAs in yeast require an internal conserved sequence (ICS) element, UACUAAC. Similar elements (ugCUAGAC) have been identified in sequences interrupting nuclear genes of the related ascomycete Neurospora crassa. To examine the structural splicing requirements in yeast, we constructed hybrid genes containing the intron of the Neurospora histone H3 gene and cloned them into high copy number yeast vectors. Subsequently we analyzed the RNAs transcribed in yeast from the fusion genes by Northern analysis and primer extended sequencing. It turned out that the Neurospora intron, which contains the sequence element UGCUAAC, can be removed, though very inefficiently, provided that it is located near the 5'-end of the primary transcript. This proves that an A at the second position of the ICS is no absolute requirement for splicing in yeast. In addition, the results indicate that the yeast splicing machinery is intron-position dependent.

Amino Acid Sequence↗

A single intronless action gene in the fission yeast Schizosaccharomyces pombe: nucleotide sequence and transcripts formed in homologous and heterologous yeast.

The actin gene of the fission yeast Schizosaccharomyces pombe has been isolated by using as a hybridization probe cloned actin DNA from the budding yeast Saccharomyces cerevisiae. In contrast to most actin genes studied from diverse eukaryotic species, the S. pombe gene is not interrupted by introns. The protein sequence deduced from the nucleotide sequence of the gene shows that the S. pombe actin is more closely related to the mammalian gamma-actin than to the actin of S. cerevisiae. Three transcripts of 1240, 1650 and 1850 nucleotides having the same 5' end but differing in the length of their 3' untranslated region are generated in the fission yeast. Only one messenger RNA of 1330 nucleotides is formed from the S. pombe actin gene in S. cerevisiae. Contrary to the observation made with other S. pombe genes transcribed in the budding yeast, the heterologous actin gene transcript is initiated 39 nucleotides upstream of the initiation start site used in the homologous yeast. The mRNA termination (or 3' processing) mechanism in the two ascomycetes also differs as the 3'end of the S. pombe actin gene transcript in S. cerevisiae does not coincide with either of the three 3'ends mapped in the fission yeast.

Actins↗

Fast light-regulated genes of Neurospora crassa.

Several physiological reactions including the sexual differentiation of the ascomycete Neurospora crassa are triggered by blue light. Mutants in the white-collar genes wc-1 and wc-2 are blind for all the blue light effects tested so far. We have previously shown that blue light induces some translatable mRNAs at different times after beginning the illumination. Here we report the cDNA cloning of four genes that are induced by blue light. Induction of these transcripts is temporally ordered (lag times from 2 to 45 min). Analysis of run-on transcripts show that the increases in mRNA levels are due to de novo transcription. None of these transcripts is inducible in white-collar mutants.

Blotting, Northern↗