A chemical reaction network for the regulation of the quinic acid (qa) gene cluster of Neurospora crassa is proposed. An efficient Monte Carlo method for walking through the parameter space of possible chemical reaction networks is developed to identify an ensemble of deterministic kinetics models with rate constants consistent with RNA and protein profiling data. This method was successful in identifying a model ensemble fitting available RNA profiling data on the qa gene cluster. With genome sequencing projects supplying an almost complete inventory of the building blocks of life, functional genomics is now facing the challenge of ''re-assembling the pieces'' (1, 2). Time-dependent mRNA (3) and protein profiling (4), protein-protein (5-8) and protein-DNA (9) interaction mapping, and the in vitro reconstruction of reaction networks (10, 11) are providing insight into the topology and kinetics of a living cell's full biochemical and gene regulatory circuitry. For the first time, it is now possible to place a particular biological circuit like those describing carbon metabolism, transcription, cell cycle, or the biological clock in simple eukaryotes in a larger context, and to examine the coupling of these circuits (12).New tools in computational biology are needed to identify these reaction networks by using well studied subcircuits like those for carbon metabolism, cell cycle, or the biological clock as a launch point into the entire circuit of a living cell. The qa gene cluster of Neurospora crassa and the GAL gene cluster of Saccharomyces cerevisiae in carbon metabolism have served as early paradigms for eukaryotic gene regulation (13,14) and are prime candidates for taking a genomic perspective on biological circuits. Mechanisms of regulation in the qa and GAL clusters with their transcriptional activator and repressors are also shared with many other regulatory networks. Because of their relative simplicity, they also provide an opportunity to test new genomic approaches to identifying chemical reaction networks or biological circuits that underlie many fundamental biological processes (15). Three opportunities exist now for identifying and refining biological circuits: the accumulation of transcriptional profiling data (3), a growing number of approaches to modeling gene regulation (11,(15)(16)(17)(18)(19)(20)(21), and the ability to carry out the in vitro reconstruction of biological circuits with a diversity of emergent properties including bistable (10) and oscillatory activity (11).However, initially, the profiling data will be scarce and the unknown parameters plentiful. Identification of the parameters in a reaction network is further complicated by the facts that the data are noisy and that our knowledge of the underlying reaction network's topology and of its participating molecular species is incomplete, even in well studied networks like those for the -switch, lac operon, trp operon, or GAL cluster. To circumvent these difficulties, we present a statistical modeling approach called the ensemble m...

An efficient transformation system has been developed for Neurospora crassa that uses spheroplasts and pVK88 plasmid DNA. pVK88 is a recombinant Escherichia coli plasmid carrying the N. crassa qa-2+ gene which encodes catabolic dehydroquinase (3-dehydroquinate hydro-lyase, EC 4.2.1.10) and is part of the qa gene cluster. The recipient strain carries a stable qa-2-mutation and an arom-9-mutation, thus lacking both catabolic and biosynthetic dehydroquinase activities. Transformants were selected as colonies able to grow in the absence of an aromatic amino acid su plement. These colonies were qa-2+ and had normal levels o catabolic dehydroquinase. DNADNA hybridization evidence with appropriate labeled probes indicates clearly that in some instances transformation involves the integration of bacterial plasmid sequences together with the qa-2+ gene into the N. crassa genome. On the basis of genetic, enzyme assay, and DNA hybridization data, at least three types of transformation events can be distinguished: (i) replacement of -the qa-2-gene by the qa-2+ gene without any effect on the expression of the other genes in the qa cluster, (ii) linked insertion of a normal qa-2+ gene accompanied by inactivation of the adjacent qa-4+ gene, and (iii) insertion of a normal qa-2+ gene at an unlinked site in the N. crassa genome. This newly integrated qa-2+ genetic material is inherited in a typical Mendelian fashion. A low level of transformation has also been obtained by using linear total N. crassa DNA. Two such qa-2+ transformants are unlinked to the qa-2-gene of the recipient. Transformation has been a well-established process for the transfer of genetic material in prokaryotes for a number of years. Until recently, such a system in higher organisms (eukaryotes) remained to be firmly established. Transformation in fungi has been limited in part because of cell wall differences which restrict DNA uptake. However, a transformation system for yeast (Saccharomyces cerevisiae) has recently been developed through the use of enzymes that partially digest the outer cell wall (1, 2). In the initial experiments described by Hinnen et al.(1), a stable leu-2-yeast strain (a double mutant strain) was transformed with a hybrid ColEl plasmid containing leu-2 + DNA. Subsequently, transformation to histidine prototrophy by using a his-3 + recombinant plasmid was also obtained (3). These transformed strains of yeast have been well characterized both for the location of the newly inserted yeast DNA and for the presence of Escherichia coli plasmid DNA sequences inserted into the yeast genome (1, 3).Although presumptive evidence for transformation of inositol-requiring strains ofNeurospora crassa has been reported (4-6), these experiments did not demonstrate the physical insertion of exogenous DNA into the N. crassa genome. By using a recently isolated recombinant E. coli plasmid (pVK88) carrying the qa-2 + gene, which encodes catabolic dehydroquinase (3-dehydroquinate hydro-lyase, EC 4.2.1.10) of N. crassa (7-9), a transformation system...

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In Neurospora crassa, five structural genes and two regulatory genes control the use of quinic acid as a carbon source. All seven genes are tightly linked to form the qa gene cluster. The entire cluster, which has been cloned and sequenced, occupies a continuous DNA segment of 17.3 kb. Three pairs of genes are divergently transcribed, including the two regulatory genes that are located at one end of the cluster and that encode an activator (qa-1F) and a repressor (qa-1S). Three of the structural genes (qa-2, qa-3, and qa-4) encode inducible enzymes that catalyze the catabolism of quinic acid. One structural gene (qa-y) encodes a quinate permease; the function of the fifth gene (qa-x) is still unclear. Present genetic and molecular evidence indicates that the qa activator and repressor proteins and the inducer quinic acid interact to control expression at the transcriptional level of all the qa genes. The activator, the product of the autoregulated qa-1F gene, binds to symmetrical 16 base pair upstream activating sequences located one or more times 5' to each of the qa genes. A conserved 28 amino acid sequence containing a six cysteine zinc binding motif located in the amino terminal region of the activator has been directly implicated in DNA binding. Evidence for other functional domains in the activator and repressor proteins are discussed. Indirect evidence suggests that the repressor is not a DNA-binding protein but forms an inactive complex with the activator in the absence of the inducer.(ABSTRACT TRUNCATED AT 250 WORDS)