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PMID: 8901135 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Identification of the native NIT2 major nitrogen regulatory protein in nuclear extracts of Neurospora crassa.

Genetica ·Vol. 97 ·No. 2 ·1996-03-00 ·Pages 153-63

Xiao X, Marzluf GA

Abstract

The nit-2 gene of Neurospora crassa encodes the major nitrogen regulatory protein which acts in a positive fashion to activate the expression of many different structural genes during conditions of nitrogen limitation. An E. coli-expressed NIT2/beta-Gal fusion protein binds specifically to DNA in vitro by recognizing GATA core elements. Nuclear extracts prepared from a wild-type N. crassa strain contain a protein factor which displays all of the properties expected for the native NIT2 protein. The native NIT2 protein in nuclear extracts binds with high affinity to DNA fragments which contain two GATA elements, weakly to fragments with a single GATA element, and fails to bind to DNAs which lack these sequences. The DNA binding ability of the protein factor in nuclear extracts is efficiently blocked by a polyclonal antibody developed against the zinc-finger region of NIT2 protein. Western blot analysis with the anti-NIT2 antiserum revealed a specific protein with a size of approximately 110,000 daltons, in excellent agreement with the predicted size of NIT2. Both the specific NIT2 DNA binding activity and the protein detected by Western blot are totally lacking in nuclear extracts of a nit-2 rip mutant strain. These results all support the conclusion that the native NIT2 protein in Neurospora cells has been identified. The NIT2 protein is localised in nuclei and could not be detected in the cytoplasmic fraction of cells subjected to nitrogen derepression or nitrogen repression, indicating that the nuclear import of NIT2 is not regulated.

MeSH Terms
Antibodies, Fungal/immunology Base Sequence Binding Sites Cell Extracts Cell Nucleus/metabolism DNA, Fungal/metabolism DNA-Binding Proteins/genetics,immunology,metabolism Fungal Proteins/genetics,metabolism Genes, Fungal Molecular Sequence Data Neurospora crassa/metabolism Nuclear Proteins/genetics,metabolism Protein Binding Transcription Factors/genetics,immunology,metabolism Zinc Fingers
Chemicals
Antibodies, Fungal Cell Extracts DNA, Fungal DNA-Binding Proteins Fungal Proteins NIT2 protein, Neurospora crassa Nuclear Proteins Transcription Factors
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Xiao X
Department of Biochemistry, Ohio State University, Columbus 43210, USA.
Marzluf G A
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Article Info
Journal
Genetica
Abbr.
Genetica
ISSN
0016-6707
DOI
10.1007/BF00054622
Published
1996-03-00
Pages
153-63
Language
English
Region
Netherlands
NLM ID
0370740
Subset
IM
Grants
NIGMS NIH HHS · GM-23367 · United States
External Links
PubMed source DOI: 10.1007/BF00054622 View journal details
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