MOLECULAR GENETICS OF SULFUR METABOLISM IN NEUROSPORA

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A major sulfur regulatory system in Neurospora crassa will be characterized at the molecular level by using a genetically- defined set of trans-acting regulatory genes and a structural gene they control. The studies are aimed at achieving an understanding of the regulatory interactions and overall organization of this multigene network. The analysis will also lead to a better understanding of how a cell regulates its sulfur status and of human inborn errors of metabolism. The system consists of the cys-3+ regulatory gene (positive regulator), three negative regulatory genes (designated scon-1+, scon-2+, scon-3+), and the arylsulfatase structural gene (ars+). The cys-3+ and ars+ genes have been have been cloned. The scon+ negative regulatory genes will be analyzed on a genetic and molecular level. Epistasis tests and function in heterokaryons will aid in establishing the hierarchy of control. At least on of the scon+ genes will be cloned by sib selection. All the cloned genes will be fully characterized (i.e., their sequence, transcripts, and gene products). Detailed studies will use the cloned genes to determine the transcriptional and post-transcriptional controls in this multigene system. Transcriptional regulation will be confirmed by nuclear transcription assays. Steady state and kinetic mRNA measurements will also be done. Posttranscriptional control will be detected by constructing gene fusions followed by gene replacement and assay. These mRNA analyses will be done with the regulatory mutants available and under different growth conditions (i.e., sulfur derepressing and repressing) so that both genetic and metabolic effects can be assessed. Potential cis- regulatory sequences will be mutagenized in vitro and re- introduced by gene replacement in order to directly confirm and test their function. The regulatory gene products will be produced in vitro and using gel mobility assays along with DNA footprinting further confirm the regulatory sequences. The interaction of cysteine (the metabolic effector) with the regulatory gene products will be examined by equilibrium dialysis. The prediction that one (or more) of the regulatory gene products will demonstrate DNA and/or cysteine binding properties will be directly tested. If time allows, the study of functional domain in the regulatory gene products will be carried out by the in vitro alteration of the genes coding sequences followed by both in vivo and in vitro assessment of function.