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W E Timberlake

Publications and source records attributed to W E Timberlake.

At least 73 records · Page 4Linked to original sources
PubMed1985

Primary structure of the trpC gene from Aspergillus nidulans.

We have determined the structure and complete nucleotide sequence of the trifunctional trpC gene from the Ascomycetous fungus Aspergillus nidulans. Results from RNA gel blot analyses showed that this gene encodes two size classes of polyribosomal, poly (A)+RNAs with approximate lengths of 2,400 and 2,600 nucleotides. S1 nuclease protection studies demonstrated that the distribution into the two size classes is due to selection of alternative sites for polyadenylation. The transcription units contain a single open translation reading frame of 2,304 nucleotides. The sequence of this reading frame is approximately 40% divergent from the sequence of the functionally analogous trp-1 gene from Neurospora crassa (Schechtman, M.G. and Yanofsky, C., J. Mol. Appl. Gen. 2:83-99). The predicted amino acid sequence of the A. nidulans trpC polypeptide is also 40% divergent from the predicted amino acid sequence of the N. crassa trp-1 polypeptide. The A. nidulans gene has considerably less bias in codon selection than observed for the N. crassa gene. Discrete regions of DNA homology were also found in similar positions in the 5' and 3' flanking sequences of the Aspergillus and Neurospora genes. Similar regions of homology were not observed in other Aspergillus or Neurospora genes that have been sequenced. Thus, if these evolutionarily conserved sequences act as signals for transcription initiation or polyadenylation, or are involved in gene regulation, their functions are restricted to a subset of protein coding genes in these two closely related fungi.

Amino Acid Sequence↗
PubMed1985

Expression of an Escherichia coli beta-galactosidase fusion gene in Aspergillus nidulans.

We inserted in frame the Escherichia coli lacZ gene into the protein-coding region of the Aspergillus nidulans trpC gene and introduced the resultant fused gene into the A. nidulans genome. A functional beta Gal fusion protein was produced. Removal of the trpC transcription and translation initiation sequences from the fusion gene abolished production of the fusion protein, showing that expression is dependent on these sequences. Thus, lacZ fusions should be of use for estimating gene activity in a. nidulans.

Aspergillus nidulans↗
PubMed1985

A cosmid for selecting genes by complementation in Aspergillus nidulans: Selection of the developmentally regulated yA locus.

We constructed a 9.9-kilobase cloning vector, designated pKBY2, for isolating genes by complementation of mutations in Aspergillus nidulans. pKBY2 contains the bacteriophage lambda cos site, to permit in vitro assembly of phage particles; a bacterial origin of replication and genes for resistance to ampicillin and chloramphenicol, to permit propagation in Escherichia coli; the A. nidulans trpC(+) gene, to permit selection in Aspergillus; and a unique BamHI restriction site, to permit insertion of DNA fragments produced by digestion with restriction endonucleases BamHI, BglII, Mbo I, or Sau3A. We used this cosmid to form a quasirandom recombinant DNA library containing 35- to 40-kilobase DNA fragments from a wild-type strain of A. nidulans. DNA from this library transformed a yellow-spored (yA(-)) pabaA(-)trpC(-)Aspergillus strain (FGSC237) to trpC(+) at frequencies of approximately 10 transformants per mug of DNA. Three of approximately 1000 trpC(+)pabaA(-) colonies obtained were putative yA(+) transformants, because they produced wild-type (green) spores. DNA from each of the green-spored transformants contained pKBY2 sequences and DNA from two transformants transduced E. coli to ampicillin resistance following treatment in vitro with a lambda packaging extract. The cosmids recovered in E. coli had similar restriction patterns and both yielded trpC(+) transformants of A. nidulans FGSC237, 85% of which produced green spores. Several lines of evidence indicate that the recovered cosmids contain a wild-type copy of the yA gene.

Journal Article↗
PubMed1985

Direct and indirect gene replacements in Aspergillus nidulans.

We performed three sets of experiments to determine whether cloned DNA fragments can be substituted for homologous regions of the Aspergillus nidulans genome by DNA-mediated transformation. A linear DNA fragment containing a heteromorphic trpC+ allele was used to transform a trpC- strain to trpC+. Blot analysis of DNA from the transformants showed that the heteromorphic allele had replaced the trpC- allele in a minority of the strains. An A. nidulans trpC+ gene was inserted into the argB+ gene, and a linear DNA fragment containing the resultant null argB allele was used to transform a trpC- argB+ strain to trpC+. Approximately 30% of the transformants were simultaneously argB-. The null argB allele had replaced the wild-type allele in a majority of these strains. The A. nidulans SpoC1 C1-C gene was modified by removal of an internal restriction fragment and introduced into a trpC- strain by transformation with a circular plasmid. A transformant containing a tandem duplication of the C1-C region separated by plasmid DNA was self-fertilized, and trpC- progeny were selected. All of these had lost the introduced plasmid DNA sequences, whereas about half had retained the modified C1-C gene and lost the wild-type copy. Thus, it is possible with A. nidulans to replace chromosomal DNA sequences with DNA fragments that have been cloned and modified in vitro by using either one- or two-step procedures similar to those developed for Saccharomyces cerevisiae.

Aspergillus nidulans↗
PubMed1984

Structure and regulated expression of the SpoC1 gene cluster from Aspergillus nidulans.

We have previously described the organization of a 13.3 kb region of the Aspergillus nidulans genome, designated SpoC1, coding for multiple poly(A)+ RNAs that accumulate in asexual spores but not in somatic cells. We have determined the limits of the SpoC1 gene cluster by investigating the transcriptional features of 53 kb of chromosomal DNA. This segment of the genome codes for at least 19 poly(A)+ RNAs, some of which are transcribed from overlapping regions. The area of developmental regulation is approximately 38 kb in length and is delimited by 1.1-kb direct repeats. With one exception, RNAs transcribed from the central part of the cluster appear late during conidiophore development and accumulate specifically in spores. The exceptional transcript appears earlier during development and accumulates specifically in cells of the conidiophore. In contrast, RNAs encoded at the borders of the cluster occur in both somatic cells and spores. The results indicate that if a chromatin-level control mechanism operates to regulate expression of the SpoC1 gene cluster, as previously suggested by us, additional levels of regulation must also exist.

Aspergillus nidulans↗
PubMed1984

Transformation of Aspergillus nidulans by using a trpC plasmid.

We constructed a chimeric plasmid carrying a complete copy of the trifunctional trpC gene from the Ascomycete fungus Aspergillus nidulans. This plasmid, designated pHY201, replicates in Escherichia coli, where it confers resistance to ampicillin and chloramphenicol and complements trpC mutants lacking phosphoribosylanthranilate isomerase activity. We used pHY201 to transform an A. nidulans trpC- strain to trpC+ at frequencies of greater than 20 stable transformants per microgram of DNA. Southern blot analysis of DNA from transformants showed that pHY201 DNA had integrated into the A. nidulans chromosomes in a majority of cases. Most of the integration events appeared to occur at the site of the trpC- allele of the recipient strain. In several instances, we succeeded in recovering pHY201, or derivatives thereof, from A. nidulans transformants by restriction endonuclease digestion of chromosomal DNA, ligation, and transformation of E. coli.

Ampicillin↗
PubMed1983

Developmental regulation of the Aspergillus nidulans trpC gene.

We have cloned the trifunctional trpC gene from Aspergillus nidulans by hybrid phage lambda complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase activity. Four different phages sharing a 4.3-kilobase region were obtained. Plasmid subclones containing this region also complemented the E. coli trpC mutant. We determined that a 1.8-kilobase DNA fragment was minimally required for complementation. The fragment hybridized with two poly(A)+ RNAs, 3.0 and 3.2 kilobases in length. We infer that these transcripts are Aspergillus trpC mRNAs and that the entire Aspergillus trpC gene is not required for complementation in E. coli. Levels of both trpC transcripts in poly(A)+ RNA are regulated by growth medium composition. They were highest when cells were grown in minimal medium containing nitrate as the nitrogen source and lowest when cells were grown in medium containing yeast extract. The concentrations of the transcripts are also regulated during conidiophore development. Conidiating cultures grown on medium containing yeast extract had significantly higher levels of both transcripts than did hyphae grown in minimal medium containing nitrate. Levels of the transcripts in mature spores were equivalent to those found in hyphae grown in minimal medium containing nitrate. Results from nutritional experiments with an A. nidulans trpC mutant suggest that developmental regulation of trpC mRNA levels may be related to a high requirement for tryptophan or a compound derived from tryptophan during conidiation.

Amino Acid Sequence↗
PubMed1982

Clustering of spore-specific genes in Aspergillus nidulans.

We have investigated the chromosomal organization of genes that are expressed specifically in the asexual spores (conidia) of the Ascomycete fungus Aspergillus nidulans, using two experimental approaches. In the first, 30 different recombinant clones, containing long nuclear DNA inserts and at least one spore-specific gene, were selected randomly. The total number of spore-specific genes present in each clone was then determined by RNA blot analysis. In the second approach, several chromosomal recombinant DNA libraries, having average insert lengths ranging from 1 to 15 kilobases, were constructed. The fraction of clones in each library having one or more spore-specific poly(A)+RNA-coding regions was then determined by colony or plaque filter hybridization with radiolabeled, spore-specific, complementary DNA. The results from these experiments were compared to statistical predictions based on the assumption that the spore-specific genes are randomly distributed in the Aspergillus genome. In both cases, the experimental values deviated significantly from the predicted values, demonstrating that the spore-specific genes are nonrandomly arranged in the genome. Rather, they appear frequently to occur in tightly linked clusters.

Aspergillus nidulans↗
PubMed1981

Organization of a gene cluster expressed specifically in the asexual spores of A. nidulans.

We have investigated the organization and regulation of a 13.3 kb region of the Aspergillus nidulans genome that is preferentially expressed during conidiophore development. This cloned DNA segment codes for six polysomal poly(A)RNAs present in dormant asexual spores (conidia) at from 8 to 50 copies per cell. The genes encoding these RNAs occur once per haploid genome, are separate and distinct, appear to be colinear with their mature RNA products and are present in both polarities. Two of the genes have short regions of homology near their 5' ends, but otherwise the segment is internally unique. All of the RNAs are absent from or present at very low levels in wild-type somatic cells (Hyphae) and developing cultures of asporogenous mutant strains. In synchronously conidiating wild-type cultures, each of the RNAs can first be detected at a time coinciding with the appearance of mature spores. Thus this region comprises a cluster of tightly linked, discrete genes, which are all expressed at the same time, but not to the same extent, in a single differentiating cell type.

Aspergillus nidulans↗
PubMed1981

Electron microscopy of Achlya deoxyribonucleic acid sequence organization.

Electron microscopic analysis of reassociated deoxyribonucleic acid (DNA) from the aquatic fungus Achlya bisexualis revealed details of the sequence arrangement of the inverted repeats and both the highly and moderately repetitive sequence clusters. We used the gene 32 protein-ethidium bromide technique for visualizing the DNA molecules, a procedure which provides excellent contrast between single- and double-stranded DNA regions. Long (greater than 6-kilobase) DNA fragments were isolated after reannealing to two different repetitive C0t values, and the renatured structures were then visualized in an electron microscope. Our results showed that the inverted repeat sequences were short (0.5 kilobase, number-average) and separated by nonhomologous DNA of various lengths. These pairs of sequences were not clustered within the genome. Both highly repetitive and moderately repetitive DNA sequences were organized as tandem arrays of precisely paired, regularly repeating units. No permuted clusters of repeating sequences were observed, nor was there evidence of interspersion of repetitive with single-copy DNA sequences in the Achlya genome.

Chytridiomycota↗
PubMed1980

Molecular cloning and selection of genes regulated in Aspergillus development.

Over 350 clones homologous to poly(A)+ RNAs that are significantly more prevalent in conidiating cultures of Aspergillus nidulans than in somatic cells have been selected from a recombinant DNA library formed between nuclear DNA and lambda Charon 4A. The procedure used for this selection involved in situ hybridization to a cDNA probe which had been selectively depleted of sequences represented in somatic cells by complement hybridization. Five of these clones have been characterized further. All but one encoded poly(A)+ RNAs that were at least ten times more prevalent in conidiating cultures than in somatic cells. One clone hybridized to a single, developmentally regulated RNA. The three others were complementary to several RNAs having different molecular weights, each of which was more prevalent in condiating cultures than in vegetative cells. These results and quantitative aspects of the selection procedure suggest that developmentally controlled poly(A)+ RNA coding regions may not be distributed randomly in the Aspergillus genome.

Aspergillus nidulans↗
PubMed1980

Developmental regulation of laccase levels in Aspergillus nidulans.

Asexual spores (conidia) of Aspergillus nidulans contain a dark green pigment which is not present in other cell types. Synthesis of this pigment is catalyzed, in part, by a developmentally controlled p-diphenol oxidase, or laccase, encoded at the gamma A genetic locus (A. J. Clutterbuck, J. Gen. Microbiol. 70:423-435, 1972). We have investigated the mechanisms regulating expression of the gamma A gene of A. nidulans. Vegetative hyphae grown in submerged culture lacked detectable laccase enzyme activity and neither contained nor synthesized immunoprecipitable laccase protein. When such cultures were induced to conidiate by harvesting the cells onto filter papers and aerating them, laccase levels began to increase after 10 to 16 h, reached a peak at 20 to 36 h, and then declined slowly. Immunological assays showed that increases in laccase enzyme activity were (i) proceded by a transient rise in the relative rate of laccase protein synthesis and (ii) closely paralleled by increases in the amount of laccase protein. Addition of cycloheximide to cultures at any time after inducing conidiation inhibited further accumulation of laccase enzyme activity. These data are most consistent with increases in laccase levels being due to regulated, de novo synthesis of laccase protein. Addition of inhibitors of ribonucleic acid synthesis to conidiating cultures also inhibited further accumulation of laccase, suggesting that laccase expression is regulated by alterations in the transcriptional activity of the gamma A locus.

Aspergillus nidulans↗
PubMed1979

Restriction endonuclease mapping by crossed contact hybridization: the ribosomal RNA genes of Achlya ambisexualis.

A rapid, convenient and economical method for the hybridization of electrophoretically resolved RNA to DNA restriction fragments immobilized on nitrocellulose filters is described. DNA was digested, electrophoresed on agarose gels in a wide band and transferred to a nitrocellulose filter. The filter was then placed on the surface of a second gel containing radioactively labeled RNA electrophoresed under denaturing conditions in a similar way. The filter and gel were oriented so that the DNA and RNA bands were perpendicular to one another and the RNA was transferred from the gel through the filter under conditions which promote RNA-DNA hybridization. Following washing, the filter was autoradiographed. RNA-DNA sequence relationships could be conveniently determined from the spots produced at regions of intersection of homologous nucleic acids. The two dimensional array formed in this procedure fascilitates the rapid ordering of DNA restriction fragments. An example of its use for this purpose is presented.

Base Sequence↗
PubMed1978

Low repetitive DNA content in Aspergillus nidulans.

DNA-DNA reassociation experiments show that the genome of Aspergillus nidulans consists of approximately 97 to 98 percent unique and 2 to 3 percent reiterated sequences. The reiterated DNA sequences have a complexity of about 11,000 base pairs and are repeated approximately 60 times per haploid genome. Ribosomal RNA-DNA hybridization experiments indicate that most of the repetitive DNA codes for ribosomal RNA.

Aspergillus nidulans↗
PubMed1978

Diversity and abundance of polyadenylated RNA from Achlya ambisexualis.

The diversity, abundance, and DNA sequence representation of poly(adenylic acid) containing RNA derived from cells of Achlya ambisexualis cultured in defined and undefined media have been determined. The kinetics of hybridization of polyadenylated RNA with complementary DNA were the same for both culture conditions and revealed the presence of three frequency classes containing 29, 220, and 3000 different sequences of an average length of 1150 nucleotides. Complexity estimates derived from experiments in which polyadenylated RNA was hybridized to unique sequence DNA were in good agreement with these results. The kinetics of hybridization of complementary DNA with an excess of nuclear DNA indicate that approximately 10% of the RNA is transcribed from reiterated DNA sequences while the remainder is transcribed from single copy sequences.

Base Sequence↗