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W E Timberlake

Publications and source records attributed to W E Timberlake.

At least 19 recordsLinked to original sources
PubMed1999

The multiply-regulated gabA gene encoding the GABA permease of Aspergillus nidulans: a score of exons.

We describe the cloning, sequence and expression of gabA, encoding the gamma-amino-n-butyrate (GABA) permease of the fungus Aspergillus nidulans. Sequence changes were determined for three up-promoter (gabI ) and six gabA loss-of-function mutations. The predicted protein contains 517 residues and shows 30.3% overall identity with a putative GABA permease of Arabidopsis thaliana, 29.6% identity with the yeast choline transporter and 23.4% identity with the yeast UGA4 GABA permease. Structural predictions favour 11-12 transmembrane domains. Comparison of the genomic and cDNA sequences shows the presence of 19 introns, an unusually large number of introns for, we believe, any fungal gene. In agreement with the wealth of genetic data available, transcript level analyses demonstrate that gabA is subject to carbon catabolite and nitrogen metabolite repression, omega-amino acid induction and regulation in response to ambient pH (being acid-expressed). In agreement with this, we report consensus binding sites 5' to the coding region, six each for CreA and AREA and one for PacC, the transcription factors mediating carbon catabolite and nitrogen metabolite repression and response to ambient pH respectively.

Amino Acid Sequence↗
PubMed1998

Increased nuclear traffic chaos in hyphae of Aspergillus nidulans: molecular characterization of apsB and in vivo observation of nuclear behaviour.

Filamentous fungi are model microorganisms for studying nuclear migration in eukaryotic cells. Two genes, apsA and apsB (=anucleate primary sterigmata), were identified in Aspergillus nidulans that affect nuclear distribution in hyphae and specifically block conidiophore development at the metula stage when mutant. Here we describe the cloning, sequencing and molecular analysis of apsB. The gene encodes a 121 kDa coiled-coil, hydrophilic protein that was localized in the cytoplasm. No protein-protein interaction was detected between ApsB and ApsA, a membrane-associated, previously identified protein. An apsB null mutant was characterized by video epifluorescence microscopy using strains that express green fluorescent protein (GFP) in nuclei. With this novel approach, we have discovered a new mutant phenotype and have found that nuclei display an increased chaotic movement in older hyphal compartments that results in clustering and an uneven distribution of these organelles. These results suggest a regulatory role of ApsB in nuclear migration.

Amino Acid Sequence↗
PubMed1997

In vitro reconstruction of the Aspergillus (= Emericella) nidulans genome.

A physical map of the 31-megabase Aspergillus nidulans genome is reported, in which 94% of 5,134 cosmids are assigned to 49 contiguous segments. The physical map is the result of a two-way ordering process, in which clones and probes were ordered simultaneously on a binary DNA/DNA hybridization matrix. Compression by elimination of redundant clones resulted in a minimal map, which is a chromosome walk. Repetitive DNA is nonrandomly dispersed in the A. nidulans genome, reminiscent of heterochromatic banding patterns of higher eukaryotes. We hypothesize gene clusters may arise by horizontal transfer and spread by transposition to explain the nonrandom pattern of repeats along chromosomes.

Aspergillus nidulans↗
PubMed1996

Wild chromosomal variants in Aspergillus nidulans.

Pulsed-field gel electrophoresis and a chromosome-specific cosmid DNA library were used to determine the karyotypes of wild-type Aspergillus nidulans isolates from around the world. Overall, little structural variation was found, with a few major exceptions. One isolate possessed a non-essential B-chromosome of about 1.0 million base pairs (mb). Another isolate had undergone a non-reciprocal translocation of about 1.6 mb of chromosome VI onto chromosome VIII. Other than these chromosomal differences, these isolates appeared phenotypically normal. To analyze its effects on meiosis, the translocation isolate was outcrossed with another wild-type derivative that had a normal electrophoretic karyotype. This cross produced a range of phenotypes, including duplicated progeny that had a barren phenotype similar to that described for Neurospora partial disomics. The duplication was somewhat vegetatively unstable. This is the first association of sterility with chromosomal duplication in A. nidulans.

Aspergillus nidulans↗
PubMed1996

On the consistency of a physical mapping method to reconstruct a chromosome in vitro.

During recent years considerable effort has been invested in creating physical maps for a variety of organisms as part of the Human Genome Project and in creating various methods for physical mapping. The statistical consistency of a physical mapping method to reconstruct a chromosome, however, has not been investigated. In this paper, we first establish that a model of physical mapping by binary fingerprinting of DNA fragments is identifiable using the key assumption-for a large randomly generated recombinant DNA library, there exists a staircase of DNA fragments across the chromosomal region of interest. Then we briefly introduce epi-convergence theory of variational analysis and transform the physical mapping problem into a constrained stochastic optimization problem. By doing so, we prove epi-convergence of the physical mapping model and epi-convergence of the physical mapping method. Combining the identifiability of our physical mapping model and the epi-convergence of a physical mapping method, finally we establish strong consistency of a physical mapping method.

Aspergillus nidulans↗
PubMed1996

Loss of meiosis in Aspergillus.

If strictly mitotic asexual fungi lack recombination, the conventional view predicts that they are recent derivatives from older meiotic lineages. We tested this by inferring phylogenetic relationships among closely related meiotic and strictly mitotic taxa with Aspergillus conidial (mitotic) states. Phylogenies were constructed by using DNA sequences from the mitochondrial small ribosomal subunit, the nuclear ribosomal internal transcribed spacers, and the nuclear 5.8S ribosomal gene. Over 920 bp of sequence was analyzed for each taxon. Phylogenetic analysis of both the mitochondrial and nuclear data sets showed at least four clades that possess both meiotic and strictly mitotic taxa. These results support the hypothesis that strictly mitotic lineages arise frequently from more ancient meiotic lineages with Aspergillus conidial states. Many of the strictly mitotic species examined retained characters that may be vestiges of a meiotic state, including the production of sclerotia, sclerotium-like structures, and hülle cells.

Aspergillus↗
PubMed1996

Saccharomyces cerevisiae TEC1 is required for pseudohyphal growth.

Diverse eukaryotic organisms share developmental transcription factors with homologous DNA-binding domains. We showed that the developmental regulator AbaA, a member of the ATTS/TEA (AbaA, TEF-1, TEC1, Scalloped/TEF-1, TEC1, AbaA) class of transcription factors of the filamentous fungus Aspergillus nidulans, induces pseudohyphal development in the yeast Saccharomyces cerevisiae. The S. cerevisiae homologue of AbaA, TEC1p, is required for this morphological transition. We provide evidence that TEC1p functions in co-operation with STE12p to induce pseudohyphal development.

Amino Acid Sequence↗
PubMed1996

catA, a new Aspergillus nidulans gene encoding a developmentally regulated catalase.

Aspergillus nidulans asexual sporulation (conidiation) is a model system for studying gene regulation and development. The CAN5 cDNA is one of several clones isolated based on transcript induction during conidiation. Here we present the molecular characterization of its corresponding gene, demonstrating that it encodes a developmentally regulated catalase, designated catA. The catA 744-amino-acid-residue polypeptide shows significant identity to other catalases. Its similarity to prokaryotic catalases is greater than to other fungal catalases. catA mRNA is barely detectable in growing mycelia, highly induced during sporulation, and present in isolated spores. However, catA expression is not dependent on the developmental regulatory genes brlA, abaA and wetA. Direct catalase activity determination in native gels revealed the existence of two bands of activity. One of these bands represented the major activity during vegetative growth and was induced during sporulation. The second catalase activity appeared after the induction of sporulation and was the predominant activity in spores. Disruption of catA abolished the major spore catalase without eliminating the vegetative activity, indicating the existence of at least two catalase genes in A. nidulans. catA-disrupted mutants produced spores that were sensitive to H2O2, as compared to wild-type spores. The increase in the activity of the vegetative catalase and the appearance of a second catalase during asexual sporulation is consistent with the occurrence of an oxidative stress during development.

Amino Acid Sequence↗
PubMed1995

Aspergillus nidulans apsA (anucleate primary sterigmata) encodes a coiled-coil protein required for nuclear positioning and completion of asexual development.

Many fungi are capable of growing by polarized cellular extension to form hyphae or by isotropic expansion to form buds. Aspergillus nidulans anucleate primary sterigmata (apsA) mutants are defective in nuclear distribution in both hyphae and in specialized, multicellular reproductive structures, called conidiophores. apsA mutations have a negligible effect on hyphal growth, unlike another class of nuclear distribution (nud) mutants. By contrast, they almost completely block entry of nuclei into primary buds, or sterigmata (bud nucleation), produced during development of conidiophores. Failure of the primary sterigmata to become nucleated results in developmental arrest and a failure to activate the transcriptional program associated with downstream developmental steps. However, occasionally in mutants a nucleus enters a primary bud and this event relieves the developmental blockage. Thus, there is a stringent developmental requirement for apsA function, but only at the stage of primary bud formation. apsA encodes a 183-kD coiled-coil protein with similarity to Saccharomyces cerevisiae NUM1p, required for nuclear migration in the budding process.

Actins↗
PubMed1995

dewA encodes a fungal hydrophobin component of the Aspergillus spore wall.

An anonymous cDNA clone, pCAN4, was shown previously to correspond to an mRNA that accumulates preferentially during asexual sporulation of the filamentous fungus Aspergillus nidulans. The peptide encoded by pCAN4 is a fungal hydrophobin, a group of small, hydrophobic cell wall proteins. When the CAN4 gene was disrupted, conidia and conidiophores appeared to be normal, but sporulating colonies wetted more rapidly with detergent solutions than did the wild type. We renamed CAN4 dewA for the detergent wettable phenotype and mapped it to chromosome V, 24 map units from cysC. The A. nidulans rodA gene also encodes a sporulation-specific fungal hydrophobin. Spores of a dewA- rodA- double mutant were less hydrophobic than those of either mutant alone, showing that dewA and rodA contribute independently to spore-wall hydrophobicity. Immunolocalization of DewA by epitope tagging demonstrated that DewA is present in the spore wall, but not in the walls of germ tubes, hyphae or cells of the spore-producing conidiophore. We conclude that dewA encodes a new fungal hydrophobin component of the conidial wall.

Amino Acid Sequence↗
PubMed1994

A fast random cost algorithm for physical mapping.

Ordering clones from a genomic library into physical maps of whole chromosomes presents a central computational/statistical problem in genetics. Here we present a physical mapping algorithm for creating ordered genomic libraries or contig maps by using a random cost approach [Berg, A. (1993) Nature (London) 361, 708-710]. This random cost algorithm is 5-10 times faster than existing physical mapping algorithms and has optimization performance comparable to existing procedures. The speedup in the algorithm makes practical the widespread use of bootstrap resampling to assess the statistical reliability of links in the physical map as well as the use of more elaborate physical mapping criteria to improve map quality. The random cost algorithm is illustrated by its application in assembling a physical map of chromosome IV from the filamentous fungus Aspergillus nidulans.

Aspergillus nidulans↗
PubMed1994

The Penicillium chrysogenum and Aspergillus nidulans wetA developmental regulatory genes are functionally equivalent.

Aspergillus nidulans and Penicillium chrysogenum are related fungi that reproduce asexually by forming multicellular conidiophores and uninucleate conidia. In A. nidulans, spore maturation is controlled by the wetA (AwetA) regulatory gene. We cloned a homologous gene (PwetA) from P. chrysogenum to determine if spore maturation is regulated by a similar mechanism in this species. The PwetA and AwetA genes are similar in structure and functional organization. The inferred polypeptides share 77% overall amino acid sequence similarity, with several regions having > 85% similarity. The genes also had significant, local sequence similarities in their 5' flanking regions, including conserved binding sites for the product of the regulatory gene abaA. PwetA fully complemented an A. nidulans wetA deletion mutation, demonstrating that PwetA and its 5' regulatory sequences function normally in A. nidulans. These results indicate that the mechanisms controlling sporulation in A. nidulans and P. chrysogenum are evolutionarily conserved.

Amino Acid Sequence↗
PubMed1994

Sexual origins of British Aspergillus nidulans isolates.

Aspergillus nidulans is a holomorphic fungus, capable of producing both meiotically and mitotically derived spores. Meiosis may be an evolutionary relic in this species because it is potentially capable of mitotic recombination and because most Aspergilli lack the ability to produce meiotic spores. We tested the null hypothesis that meiosis has been a major factor in the origin of strains of A. nidulans from Great Britain by estimating linkage disequilibrium among restriction fragment length polymorphisms. These strains belong to different heterokaryon compatibility groups and are thus incapable of undergoing mitotic recombination with one another, so any recombination evidenced by linkage equilibrium is assumed to be the result of meiosis. Eleven cosmid clones of known chromosomal origin were used to generate multilocus genotypes based on restriction-pattern differences for each heterokaryon compatibility group. Low levels of genetic variation and little linkage disequilibrium were found, indicating that the heterokaryon compatibility groups represent recently diverged lineages that arose via meiotic recombination. The null hypothesis that loci are independent could not be rejected. Additionally, low levels of electrophoretic karyotype variation were indicative of meiosis. We conclude that although A. nidulans probably propagates in a primarily clonal fashion, recombination events are frequent enough to disrupt the stable maintenance of clonal genotypes. We further conclude that the British heterokaryon compatibility groups arose via recombination and not through novel mutation.

Aspergillus nidulans↗
PubMed1994

ODS_BOOTSTRAP: assessing the statistical reliability of physical maps by bootstrap resampling.

In the program ODS_BOOTSTRAP we provide a methodology for quickly ordering clones in a genomic library into a physical map and for applying a statistical tool known as the bootstrap to assess the statistical reliability of a clonal ordering. Each clone is assigned a binary fingerprint by one of a variety of experimental approaches to physical mapping. For example, the binary fingerprints might be generated by hybridizing a panel of m probes to a library of n clones. The resulting n x m binary data matrix, X, is input to ODS_BOOTSTRAP, which utilizes the similarity in binary fingerprints of clones to construct a physical map. Under this particular implementation of bootstrap resampling, the m probes (or columns of the data matrix) are sampled randomly with replacement in the computer to generate a new n x m data matrix, X*, from which a second physical map is constructed. The resampling process is repeated 100 or more times to generate 100 or more X* matrices. The resulting 100 or more physical maps are compared with the original physical map based on the original data matrix X by counting how often links in the original physical map reappear. Three confidence statistics are introduced for each link in a physical map. The statistic C1 is defined as the percentage of time two neighboring clones on the original map reappear as neighbors under resampling. The statistic C2 is defined as the percentage of time that two neighboring clones i and j on the original map reappear as neighbors or that a clone with an identical binary fingerprint to clone i reappears as a neighbor to clone j. The statistic C3 is defined as the percentage of time that two neighboring clones on the original map reappear in the same contig under resampling.

Algorithms↗
PubMed1994

The Aspergillus nidulans abaA gene encodes a transcriptional activator that acts as a genetic switch to control development.

The Aspergillus nidulans abaA gene encodes a protein containing an ATTS DNA-binding motif and is required for the terminal stages of conidiophore development. Results from gel mobility shift and protection, missing-contact, and interference footprint assays showed that AbaA binds to the sequence 5'-CATTCY-3', where Y is a pyrimidine, making both major- and minor-groove contacts. Multiple AbaA binding sites are present in the cis-acting regulatory regions of several developmentally controlled structural genes as well as those of the upstream regulatory gene brlA, the downstream regulatory gene wetA, and abaA itself. These cis-acting regulatory regions confer AbaA-dependent transcriptional activation in a heterologous Saccharomyces cerevisiae gene expression system. From these observations, we propose that the AbaA transcription factor establishes a novel set of feedback regulatory loops responsible for determination of conidiophore development.

Base Sequence↗