A gene cluster in Nuerospora crassa coding for an aggregate of five aromatic synthetic enzymes.

Giles, Norman H.; Case, Mary E.; Partridge, Chris; Ahmed, S. I.

doi:10.1073/pnas.58.4.1453

Cited by 115 publications

(52 citation statements)

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“…One remarkable feature of the pathway is the diversity of organisation of the enzymes and the genes encoding them in a wide variety of species. The pentafunctional arom enzyme represents one extreme-one gene, five activities [1,2]; at the opposite extreme, Escherichia coli has five widely scattered genes [3], from which five separable 2.3. Isolation of RNA 1 ml samples of an exponentially growing S. cerevisiae culture were harvested by centrifugation (microfuge, 1 min) and resuspended in 200/zl of 200 mM Tris-HC1 (pH 7.5), 0.5 M NaCI, 10 mM EDTA.…”

Section: Introductionmentioning

confidence: 99%

The Saccharomyces cerevisiae ARO1 gene An example of the co‐ordinate regulation of five enzymes on a single biosynthetic pathway

Duncan

Edwards²,

Coggins

1988

FEBS Letters

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The ARO1 gene of Saccharomyces cerevisiae encodes the arom multifunctional enzyme. Specific inhibitors of amino acid biosynthesis have been used to obtain evidence that expression of a cloned ARO1 gene is regulated in response to amino acid limitation. Northern blot analysis and sequence studies indicate that ARO1 is regulated by the well characterised S. cerevisiae 'general control' mechanism. This provides a very economical means of simultaneously tailoring the synthesis of five shikimate pathway enzymes to the needs of the cell.

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Section: Introductionmentioning

confidence: 99%

The Saccharomyces cerevisiae ARO1 gene An example of the co‐ordinate regulation of five enzymes on a single biosynthetic pathway

Duncan

Edwards²,

Coggins

1988

FEBS Letters

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“…The small possibility that during exponential growth the mycelia were preferentially using the 20% H20 and thus synthesized no heavily labeled protein was also eliminated. The constitutively synthesized arom multienzyme aggregate (18) was isolated from 18-hr cultures grown in 100% H20 and in 80% D20 plus 20% H20. The density was found to increase from 1.2720 to 1.3180 o./ 12. g/cm3 , which demonstrates that D20 is utilized during exponential growth.…”

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confidence: 99%

Proof of de novo synthesis of the qa enzymes of Neurospora crassa during induction

Reinert

Giles

1977

Proc. Natl. Acad. Sci. U.S.A.

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In Neurospora crassa three inducible enzymes are necessary to catabolize quinic acid to protocatechuic acid. The three genes encoding these enzymes are tightly linked on chromosome VII near methionine-7 (me-7). This qa cluster includes a fourth gene, qa-1, which encodes a regulatory protein apparently exerting positive control over transcription of the other three qa genes. However, an alternative hypothesis is that the qa-I protein simply activates preformed polypeptides derived from the three structural genes. The use of density labeling with D20 demonstrated conclusively that the qa enzymes are synthesized de novo only during induction on quinic acid. Native catabolic dehydroquinase (5-dehydroquinate dehydratase; 5-dehydroquinate hydro-lyase, EC 4.2.1.10) (a homopolymer of ca 22 identical subunits) has a density of 1.2790 g/cm3 as determined by centrifugation in a modified cesium chloride density gradient. Growth in H20 followed by induction in 95% D2O shifts the density of the enzyme to 1.3130 g/cm3, indicating de novo synthesis during induction. In the reciprocal experiment, i.e., growth in 80% D20 followed by induction in either 95% D20 or H20, the densities of catabolic dehydroquinase were 1.3135 and 1.2800 g/cm3, respectively. Because growth on D20 does not affect the density of the H20-induced enzyme, there can be no significant synthesis of catabolic dehydroquinase prior to induction. Similar results were obtained for a second qa enzyme, quinate dehydrogenase (quinate:NAD+ oxidoreductase, EC 1.1.1.24). Thus, induction of two qa enzymes involves de nQvo protein synthesis, not enzyme activation or assembly. In Neurospora crassa, the catabolism of quinic acid to acetate for use as a carbon source has been amply studied (1-3). Recent interest has centered on the initial three inducible catabolic enzymes necessary to convert quinic acid to protocatechuic acid (4-13). These enzymes are not physically associated with one another, yet the three genes encoding them are tightly linked on chromosome VII near me-7. In Giles, unpublished). This qa gene cluster also includes a fourth gene, qa-1, which apparently regulates the transcription of the three other genes in the cluster.There is strong genetic evidence that the qa-1 gene encodes a regulatory protein which, in the presence of quinic acid, exerts positive control over the synthesis of the enzymes encoded in the three adjacent structural genes (6, 10). The appearance of enzyme activity is very specifically regulated. In the presence of a preferred carbon source, e.g., sucrose, the activities of all three enzymes are quite low. Quinate as a sole carbon source causes a coordinate induction of all three enzymes (9). This increase in enzyme activity can be inhibited by the simultaneous addition of cycloheximide to the medium (ref. 5; J. A.Hautala, unpublished data; this paper). This effect strongly suggests that all three enzymes are being synthesized de novo during induction. An alternate explanation, which as yet has never been conclusively eliminate...

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“…It is likely that this complex locus corresponds to the arom gene cluster of Neurospora crassa (Giles et al, 1967) which codes for an enzyme complex bearing enzyme activity for steps two to six in early aromatic amino acid biosynthesis.…”

Section: Discussionmentioning

confidence: 99%

Isolation and Genetical Classification of Aromatic Amino Acid Auxotrophic Mutants in Schizosaccharomyces pombe

Strauss

1979

Journal of General Microbiology

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173Mutants of Schizosaccharomyces pombe with single and multiple requirements for aromatic amino acids were isolated and allocated to 15 loci. Difficulties in cultivation of many of these auxotrophs were largely overcome by using a minimal medium with L-glutamic acid as sole nitrogen source. The locus-typical growth factor requirements of mutants allowed a preliminary allocation of genes to reaction sequences in aromatic amino acid biosynthesis. Several explanations for the occurrence of mutants with locus-atypical growth factor requirements are discussed. I N T R O D U C T l O NWe have attempted to isolate mutants of Schizosaccharomyces pombe auxotrophic for aromatic amino acids and aromatic vitamins. Mutants blocked in enzymes of the tryptophan-synthesizing branch of the pathway (Schweingruber & Dietrich, 1973) and others defective in isoenzymes controlling the first step of aromatic amino acid biosynthesis (Schweingruber & Wyssling, 1974) have already been isolated and characterized biochemically, but no mutants with multiple requirements for aromatic amino acids, as have been described in Neurospora crassa (Giles et al., 19671, Saccharomyces cerevisiae (De Leeuw, 1968) and Aspergillus nidulans (Roberts, 1969), have been isolated. Here we report on the isolation and genetical classification of such 5. pombe mutants as well as of others with single requirements for phenylalanine, tyrosine, tryptophan or p-aminobenzoic acid. METHODS Strains.The following strains of Schizosaccharomyces pombe stem from the silica gel stock collection of Professor U. Leupold, Institute of General Microbiology, University of Bern, Switzerland : wild-type strains 972 h-and 975 h+; tryptophan auxotrophic strains, originally isolated by Dietrich (1970), trpIA-BI h-/h+, trpIB-4 h-/h+, trpIC-3 h-/h+, trp2-2 h-/h+, trp3-23 h-/h+ and trp4-47 h-/h+.Media. Liquid yeast extract medium (YEL), malt extract agar (MEA) and minimal agar (MMA) were as described by Gutz et al. (1974). A slightly modified minimal medium (MMAg) was found to be more suitable for cultivation of aromatic amino acid auxotrophic mutants: in this, L-glutamic acid (10 g 1-1) replaced ammonium sulphate, Difco agar was used and the pH was adjusted to 5.5. Supplemented media (designated ' + aro') contained L-phenylalanine, L-tyrosine and L-tryptophan (all at M) and p-aminobenzoic acid M), added to the medium after autoclaving. Plates with supplemented media were stored at 4 "C and were used within 2 weeks.

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A gene cluster in Nuerospora crassa coding for an aggregate of five aromatic synthetic enzymes.

Cited by 115 publications

References 12 publications

The Saccharomyces cerevisiae ARO1 gene An example of the co‐ordinate regulation of five enzymes on a single biosynthetic pathway

The Saccharomyces cerevisiae ARO1 gene An example of the co‐ordinate regulation of five enzymes on a single biosynthetic pathway

Proof of de novo synthesis of the qa enzymes of Neurospora crassa during induction

Isolation and Genetical Classification of Aromatic Amino Acid Auxotrophic Mutants in Schizosaccharomyces pombe

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