The goal of the proposed research is to gain a complete understanding at the molecular level of two complex regulatory circuits, one controls nitrogen metabolism, the second, sulfur metabolism in Neurospora crassa. Our laboratory has cloned, characterized, and sequenced the major regulatory genes and representative structural genes from each of these circuits. These regulatory genes encode DNA-binding proteins which are homologous to growth control or oncogene proteins of man, NIT2 being related to GF1 which controls globin gene expression in man, CYS3 has similarities with oncogene FOS. Thus, these genetically-defined control proteins can serve as excellent models to study regulatory interactions involving these human factors. Research is proposed to investigate the domain structure of three different genetically-defined control proteins can serve as excellent models to study regulatory interactions involving these human factors. Research is proposed to investigate the domain structure of three different genetically-defined regulatory proteins (NIT2 and NIT4 of the N-circuit, CYS3 in the S-circuit) in order to understand their (a) sequence-specific DNA binding, (b) metabolic control of their activity, and (c) the manner in which they mediate positive trans- activation of structural genes. Domains responsible for trans-activation will be identified by deletion analysis, and then refined with smaller deletions and single amino acid replacements. The NIT4 protein will be expressed and in vitro for each of the three regulatory proteins, NIT2, NIT4, and CYS3, will be examined to determine whether they function in gene regulation in vivo. Research is proposed to determine how changes in amino acids within the DNA binding domain of NIT2 can yield altered proteins which still efficiently bind DNA but which recognizes new nucleotide sequences. The cys-3 regulatory gene appears to be controlled much like oncogene fos and indeed to control its own expression; gene fusion experiments are planned to directly test whether autogenous control of CYS3 is an important feature of the sulfur regulatory circuit.
Showing the most recent 10 out of 69 publications
