The overall objective is to understand at the molecular level the regulation of a particular genetic system in the lower eukaryote Neurospora crassa which may serve as a model for understanding gene organization and regulation in eukaryotes in general. This system involves the qa gene cluster which encodes the inducible enzymes catalyzing the early reactions in the quinic acid catabolic pathway. The entire qa gene cluster has been cloned on a 17.6 kb fragment and sequenced. Present genetic, biochemical, and molecular evidence indicates that this cluster comprises five structural and two regulatory genes. The two regulatory genes (qa-1S and qa-1F) encode, respectively, a repressor and an activator protein. The actions of these two regulatory proteins, plus that of the inducer, quinic acid, control the synthesis of the inducible enzymes (encoded by the five structural genes) which catalyze the utilization of quinic acid as a carbon source by N. crassa. The four major specific aims of the present proposal are: (1) To identify the molecular target of the activator protein, which plays a positive regulatory role in facilitating the transcription by RNA polymerase II of all qa genes, including itself (autoregulation). Present data suggest that this target may be a 16 bp consensus sequence with dyad symmetry (i.e., an upstream activator sequence - UASQ) located 5' to each qa gene. This sequence can be deleted or modified both in vivo and in vitro and its significance studied using transformation procedures. (2) To identify the molecular target of the repressor protein. Present data suggest that this target may be the activator protein, and attempts will be made to isolate constitutive qa-1F mutants (qa-1Fc) in the coding region of the qa-1F gene, whose detection would support this hypothesis. Also additional repressor mutants will be cloned and sequenced to identify specific functional repressor domains. (3) To isolate significant amounts of both activator and repressor proteins by cloning the qa-1F and qa-1S genes into high expression vectors. These isolated proteins would be characterized by their DNA and protein binding characteristics and would be used in an available homologous N. crassa polymerase II transcription system. (4) To identify the presently unknown functions of the qa-x and qa-y genes in the qa gene cluster utilizing gene inactivation and transformation procedures.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM028777-08
Application #
3276074
Study Section
Genetics Study Section (GEN)
Project Start
1981-07-01
Project End
1991-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
8
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Georgia
Department
Type
Schools of Arts and Sciences
DUNS #
City
Athens
State
GA
Country
United States
Zip Code
30602
Crawford, J M; Geever, R F; Asch, D K et al. (1992) Sequence and characterization of the met-7 gene of Neurospora crassa. Gene 111:265-6
Case, M E; Geever, R F; Asch, D K (1992) Use of gene replacement transformation to elucidate gene function in the qa gene cluster of Neurospora crassa. Genetics 130:729-36
Asch, D K; Orejas, M; Geever, R F et al. (1991) Comparative studies of the quinic acid (qa) cluster in several Neurospora species with special emphasis on the qa-x-qa-2 intergenic region. Mol Gen Genet 230:337-44
Giles, N H; Geever, R F; Asch, D K et al. (1991) The Wilhelmine E. Key 1989 invitational lecture. Organization and regulation of the qa (quinic acid) genes in Neurospora crassa and other fungi. J Hered 82:1-7
Hiett, K L; Case, M E (1990) Induced expression of the Aspergillus nidulans QUTE gene introduced by transformation into Neurospora crassa. Mol Gen Genet 222:201-5
Avalos, J; Geever, R F; Case, M E (1989) Bialaphos resistance as a dominant selectable marker in Neurospora crassa. Curr Genet 16:369-72
Geever, R F; Huiet, L; Baum, J A et al. (1989) DNA sequence, organization and regulation of the qa gene cluster of Neurospora crassa. J Mol Biol 207:15-34
Baum, J A; Geever, R; Giles, N H (1987) Expression of qa-1F activator protein: identification of upstream binding sites in the qa gene cluster and localization of the DNA-binding domain. Mol Cell Biol 7:1256-66
Tyler, B M (1987) Transcription of Neurospora crassa 5 S rRNA genes requires a TATA box and three internal elements. J Mol Biol 196:801-11
Case, M E (1986) Genetical and molecular analyses of qa-2 transformants in Neurospora crassa. Genetics 113:569-87

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