The goal of the proposed research is to gain a complete understanding at the molecular level of two complex regulatory circuits which control nitrogen metabolism and sulfur metabolism in Neurospora crassa. The major nitrogen regulatory gene, nit-2, and the major sulfur regulatory gene, cys-3, have already been cloned; these two control genes will be completely characterized and sequenced; the regulatory protein which each encodes will be identified and sufficient amounts prepared for detailed in vitro analysis. Gene fusion studies will be employed to investigate whether the expression of these control genes is constitutive or regulated, and to determine whether amino terminal sequences of either protein allows it to be targeted to the nucleus. Shotgun transformation will be used to isolate several specific structural genes of the nitrogen and the sulfur control circuits, including several, such as the nitrate reductase gene, which are regulated by multiple control genes and effectors. Potential cis-acting control elements that serve as recognition sites for trans-acting proteins will be identified, and their roles established by in vitro mutagenesis followed by assessing their function in vivo after transformation. DNA footprinting studies both in vitro and in vivo will be carried out to determine whether the sites in the 5' flanking DNA of the structural genes defined by the mutational analysis actually serve as binding sites for the respective regulatory protein. An in vitro transcriptional system will be developed in which the transcription of cloned genes of the S or N circuit can be analyzed and the effect of the addition of purified regulatory proteins and small molecular weight effectors can be elucidated.
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