Clive F. Roberts

Molecular organisation of the quinic acid utilization (QUT) gene cluster in Aspergillus nidulans

SummaryThe functional integrity of the QUTB gene (encoding quinate dehydrogenase) has been confirmed by transformation of a qutB mutant strain. The DNA sequence of the contiguous genes QUTD (quinate permease), QUTB and QUTG (function unknown) has been determined and analysed, together with that of QUTE (catabolic 3-dehydroquinase). The QUTB sequence shows significant homology with the shikimate dehydrogenase function of the complex AROM locus of Aspergillus nidulans, and with the QA-3 quinate dehydrogenase and QA-1S (repressor) genes of Neurospora crassa. The QUTD gene shows strong homology with the N. crassa QA-Y gene and QUTG with the QA-X gene. QUTD, QUTB, and QUTG, QUTE form two pairs of divergently transcribed genes, and conserved sequence motifs identified in the two common 5′ non-coding regions show significant homology with UASGAL and UASQA sequences of the Saccharomyces cerevisiae and N. crassa Gal and QA systems. In addition, conserved 5′ sequences homologous to the mammalian CAAT box are noted and a previously unreported conserved 22 nucleotide motif is presented.

Transcription and processing signals in the 3-phosphoglycerate kinase (PGK) gene from Aspergillus nidulans.

The 3-phosphoglycerate kinase gene from Aspergillus nidulans contains two 57-bp introns and codes for a 421-amino acid (aa) protein with considerable homology to the Saccharomyces cerevisiae (68%) and mammalian (64%) proteins. Almost total conservation is found in Aspergillus of residues thought to be important to the structure and function of the yeast enzyme, and the introns fall between coding sequences for postulated structures in the N-domain. The strong codon preference found is more similar to that in other filamentous fungi than in yeast. The transcription start point (+1) has been mapped 32 bp upstream from the start codon, and the promoter region contains potential homologies for CAAT (-80 bp) and TATA (-30 bp) sequences, and certain other features common to other highly expressed genes in ascomycetes. There are three major termini 23, 83 and 115 bp beyond the stop codon and two of these are preceded by the polyadenylation consensus sequence and contain potential secondary structure.

Genetic Regulation of the Quinic Acid Utilization (QUT) Gene Cluster in Aspergillus nidulans

A large number of quinic acid non-utilizing qut mutants of Aspergillus nidulans deficient in the induction of all three quinic acid specific enzymes have been analysed. One class the qutD mutants, are all recessive and are non-inducible at pH 6.5 due to inferred deficiency in a quinate ion permease. Two regulatory genes have been identified. The QUTA gene encodes an activator protein since most qutA mutants are recessive and non-inducible although a few fully dominant mutants have been found. The QUTR gene encodes a repressor protein since recessive mutations are constitutive for all three enzyme activities. Rare dominant non-inducible mutants which revert readily to yield a high proportion of constitutive strains are inferred to be qutR mutants defective in binding the inducer. The gene cluster has been mapped in the right arm of chromosome VIII in the order: centromere - greater than 50 map units - ornB - 12 map units - qutC (dehydratase)-0.8 map units-qutD (permease), qutB (dehydrogenase), qutE (dehydroquinase), qutA (activator)-4.4 map units - qutR (repressor)-20 map units - galG. This organization differs from that of the qa gene cluster in Neurospora crassa, particularly in the displacement of qutC and qutR.

The QUTA activator and QUTR repressor proteins of Aspergillus nidulans interact to regulate transcription of the quinate utilization pathway genes

Genetic evidence suggests that the activity of the native QUTA transcription activator protein is negated by the action of the QUTR transcription repressor protein. When Aspergillus nidulans was transformed with plasmids containing the wild-type qutA gene, transformants that constitutively expressed the quinate pathway enzymes were isolated. The constitutive phenotype of these transformants was associated with an increased copy number of the transforming qutA gene and elevated qutA mRNA levels. Conversely, when A. nidulans was transformed with plasmids containing the qutR gene under the control of the constitutive pgk promoter, transformants with a super-repressed phenotype (unable to utilize quinate as a carbon source) were isolated. The super-repressed phenotype of these transformants was associated with an increased copy number of the transforming qutR gene and elevated qutR mRNA levels. These copy-number-dependent phenotypes argue that the levels of the QUTA and QUTR proteins were elevated in the high-copy-number transformants. When diploid strains were formed by combining haploid strains that contained high copy numbers of either the qutA gene (constitutive phenotype) or the qutR gene (super-repressing; non-inducible phenotype), the resulting diploid phenotype was one of quinate-inducible production of the quinate pathway enzymes, in a manner similar to wild-type. The simplest interpretation of these observations is that the QUTR repressor protein mediates its repressing activity through a direct interaction with the QUTA activator protein. Other possible interpretations are discussed in the text. Experiments in which truncated versions of the QUTA protein were produced in the presence of a wild-type QUTA protein indicate that the QUTR repressor protein recognizes and binds to the C-terminal half of the QUTA activator protein.

Spatial and biological characterisation of the complete quinic acid utilisation gene cluster in Aspergillus nidulans

SummaryHeterologous probing of restriction digests of chromosomal DNA from Aspergillus nidulans with radioactively labelled probes encoding dehydroshikimate dehydratase (QA-4) and a repressor gene (QAI-S) from Neurospora crassa revealed a pattern of hybridisation inconsistent with an equivalent single copy of each gene in A. nidulans. Screening of size-selected and total genome A. nidulans DNA libraries allowed the isolation of four unique classes of sequence, two of which hybridised to the QA-4 probe, and two of which hybridised to the QA1-S probe. In each case, one of each pair of unique sequences was able to complement the equivalent mutations qutC (=QA-4) and qutR (=QA1-S) in A. nidulans, whereas the second of each pair was unable to complement the same mutation. The complementing sequences were physically mapped relative to the previously cloned A. nidulans QUT gene cluster, demonstrating that QUTR is distal and divergently transcribed from QUTA with approximately 3.6 kb between the ATG translational start codons, and that QUTC is transcribed in the same direction as QUTD on the other side of the cluster, approximately 1.65 kb downstream of the QUTD TAA translational stop signal. The physical and genetic maps of the QUT gene cluster correlate precisely. The non-complementing A. nidulans DNA sequences that hybridise to the N. crassa QA-4 (=QUTC) and QA1-S (=QUTR) fulfill many of the criteria characteristic of pseudogenes. The derived protein sequence of the QUTG gene shows a striking similarity to the protein sequence of bovine myo-inositol monophosphatase, indicating that they evolved from a common ancestor, and suggests a role for the QUTG gene, for which no function has previously been discovered, in expression of the QUT gene cluster.

Cloning and characterization of the three enzyme structural genes QUTB, QUTC and QUTE from the quinic acid utilization gene cluster in Aspergillus nidulans

SummaryHeterologous DNA probes from the quinic acid gene cluster (QA) in Neurospora crassa (Schweizer 1981) have been used to isolate the corresponding gene cluster (QUT) from Aspergillus nidulans cloned in a phage λ vector. N. crassa probes for each of the three enzyme structural genes in the cluster have been used to identify the corresponding genes within the A. nidulans cloned DNA. The three genes are in the same relative sequence [dehydrogenase (1), QA-3 ≡ QUTB; dehydratase (3), QA-4 ≡ QUTC; dehydroquinase (2), QA-2 = QUTE] though contained within a 3.4 kb DNA sequence in Aspergillus compared to a 5.4 kb sequence in Neurospora.The A. nidulans dehydroquinase (2) gene QUTE has been shown to complement an auxotrophic mutantaro D6 of Escherichia coli lacking biosynthetic dehydroquinase when tested for growth at 30 °C.A mutant of A. nidulans lacking catabolic dehydroquinase (2) and designated qutE208 has been isolated and shown to be tightly linked to the gene cluster, which maps between the ornB and fwA loci in linkage group VIII.

Molecular cloning of the 3-phosphoglycerate kinase (PGK) gene from Aspergillus nidulans

SummaryThe Aspergillus nidulans 3-phosphoglycerate kinase gene (PGK) has been isolated from a phage λ genomic library, using the equivalent yeast gene as a hybridization probe. The location of the PGK gene within the cloned DNA has been physically mapped. The DNA sequence of a small region of the putative PGK has been determined and found to code for amino acids corresponding to the N-terminal end of the PGK protein. In contrast to the yeast PGK gene the Aspergillus gene contains a 57 base pair intron occurring between the coding sequences for amino acid 22 and 23.A DNA fragment encompassing the PGK gene was shown to hybridize a 1,700 base poly(A) mRNA, sufficient to encode the PGK polypeptide.

Cold-sensitive mutants in Aspergillus nidulans

SummaryMutant strains of Aspergillus nidulans have been isolated which grow normally on minimal medium at 37°C but not at 20°C. Growth tests indicate that these seventy-five mutant strains (designated CS, cold-sensitive) have a range of defects. Five are auxotrophic at 20°C, one (CS13) requiring isoleucine and another (CS48) choline. Many mutants are osmotic remedials. Some CS strains have altered properties at 37°C, including deoxycholate-sensitivity, actidione-resistance or actidione-ultrasensitivity.The majority of thirty-two CS strains tested segregate cold-sensitivity as single gene mutations in crosses with wild-type. Cold-sensitivity of one strain (CS67) is cytoplasmically inherited. Dominance tests in heterokaryons showed that in each of twenty-six CS strains examined cold-sensitivity is determined by recessive mutations (designated cs). Complementation analysis of nine cs mutations showed that they each affect a different function. Crosses between some cs mutants, and the allocation of other cs mutations to different linkage groups, demonstrate that mutations to cold-sensitivity are not restricted to a limited region of the genome.These results indicate that in Aspergillus nidulans selection for cold-sensitivity provides an enrichment for mutants with alterations in many different cellular properties.

Cytoplasmic inheritance of a cold-sensitive mutant in Aspergillus nidulans.

SUMMARY: Cold-sensitive mutants of bacteria are a source of strains defective in the assembly of ribosomes (Guthrie, Nashimoto & Nomura, 1969; Tai, Kessler & Ingraham, 1969). A study of cold-sensitive mutants in Aspergillus nidulans was undertaken in the hope that it would provide ribosomal mutants. Cold-sensitive (cs) mutants were isolated, and when crossed with wild-type the majority of 32 mutants tested segregated as single-gene mutations. One mutant, cs67, showed non-Mendelian segregation of its cold-sensitive character in such a cross. This paper presents further results demonstrating the extranuclear inheritance of CS67.

Genesis of eukaryotic transcriptional activator and repressor proteins by splitting a multidomain anabolic enzyme

The genes necessary for the correctly regulated catabolism of quinate in Aspergillus nidulans and Neurospora crassa are controlled at the level of transcription by a DNA-binding activator protein and a repressor protein that directly interact with one another. The repressor protein is homologous throughout its length with the three C-terminal domains of a pentafunctional enzyme catalysing five consecutive steps in the related anabolic shikimate pathway. We now report that the activator protein is homologous to the two N-terminal domains of the same pentafunctional enzyme and that this proposed structural similarity suggests a molecular mechanism by which the repressor recognises the activator protein. We believe that this is the first report of the genesis of a pair of interacting eukaryotic regulatory proteins by the splitting of a multidomain anabolic enzyme. The recruitment of preformed enzymatically active domains to a regulatory role may represent a general mechanism for the evolution of pathway-specific regulator proteins in dispensable pathways.