scholarly journals Analysis of multiple forms of nuclear factor I in human and murine cell lines.

1990 ◽  
Vol 10 (3) ◽  
pp. 1041-1048 ◽  
Author(s):  
N Goyal ◽  
J Knox ◽  
R M Gronostajski
Keyword(s):  
Cell Lines ◽  
Binding Activity ◽  
Cross Linking ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Dna Complexes ◽  
Murine Cell ◽  
Growth State ◽  
Factor I ◽  
Nuclear Factor I

Nuclear factor I (NFI) is a group of related site-specific DNA-binding proteins that function in adenovirus DNA replication and cellular RNA metabolism. We have measured both the levels and forms of NFI that interact with a well-characterized 26-base-pair NFI-binding site. Five different NFI-DNA complexes were seen in HeLa nuclear extracts by using a gel mobility shift (GMS) assay. In addition, at least six forms of NFI were shown to cross-link directly to DNA by using a UV cross-linking assay. The distinct GMS complexes detected were composed of different subspecies of NFI polypeptides as assayed by UV cross-linking. Different murine cell lines possessed varying levels and forms of NFI binding activity, as judged by nitrocellulose filter binding and GMS assays. The growth state of NIH 3T3 cells affected both the types of NFI-DNA complexes seen in a GMS assay and the forms of the protein detected by UV cross-linking.

Analysis of multiple forms of nuclear factor I in human and murine cell lines

1990 ◽  
Vol 10 (3) ◽  
pp. 1041-1048
Author(s):  
N Goyal ◽  
J Knox ◽  
R M Gronostajski
Keyword(s):  
Cell Lines ◽  
Binding Activity ◽  
Cross Linking ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Dna Complexes ◽  
Murine Cell ◽  
Growth State ◽  
Factor I ◽  
Nuclear Factor I

Nuclear factor I (NFI) is a group of related site-specific DNA-binding proteins that function in adenovirus DNA replication and cellular RNA metabolism. We have measured both the levels and forms of NFI that interact with a well-characterized 26-base-pair NFI-binding site. Five different NFI-DNA complexes were seen in HeLa nuclear extracts by using a gel mobility shift (GMS) assay. In addition, at least six forms of NFI were shown to cross-link directly to DNA by using a UV cross-linking assay. The distinct GMS complexes detected were composed of different subspecies of NFI polypeptides as assayed by UV cross-linking. Different murine cell lines possessed varying levels and forms of NFI binding activity, as judged by nitrocellulose filter binding and GMS assays. The growth state of NIH 3T3 cells affected both the types of NFI-DNA complexes seen in a GMS assay and the forms of the protein detected by UV cross-linking.

scholarly journals Calcineurin Regulates Nuclear Factor I Dephosphorylation and Activity in Malignant Glioma Cell Lines

2013 ◽  
Vol 288 (33) ◽  
pp. 24104-24115 ◽  
Author(s):  
Miranda Brun ◽  
Darryl D. Glubrecht ◽  
Shairaz Baksh ◽  
Roseline Godbout
Keyword(s):  
Malignant Glioma ◽  
Cell Lines ◽  
Glioma Cell ◽  
Nuclear Factor ◽  
Factor I ◽  
Glioma Cell Lines ◽  
Nuclear Factor I

scholarly journals Transcription factors nuclear factor I and Sp1 interact with the murine collagen alpha 1 (I) promoter.

1991 ◽  
Vol 11 (8) ◽  
pp. 4065-4073 ◽  
Author(s):  
M C Nehls ◽  
R A Rippe ◽  
L Veloz ◽  
D A Brenner
Keyword(s):  
Binding Sites ◽  
Direct Repeat ◽  
Dnase I ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Factor I ◽  
3T3 Fibroblasts ◽  
Nuclear Factor I ◽  
Simplex Virus ◽  
Nih 3T3

The collagen alpha 1(I) promoter, which is efficiently transcribed in NIH 3T3 fibroblasts, contains four binding sites for trans-acting factors, as demonstrated by DNase I protection assays (D. A. Brenner, R. A. Rippe, and L. Veloz, Nucleic Acids Res. 17:6055-6064, 1989). This study characterizes the DNA-binding proteins that interact with the two proximal footprinted regions, both of which contain a reverse CCAAT box and a G + C-rich 12-bp direct repeat. Analysis by DNase I protection assays, mobility shift assays, competition with specific oligonucleotides, binding with recombinant proteins, and reactions with specific antisera showed that the transcriptional factors nuclear factor I (NF-I) and Sp1 bind to these two footprinted regions. Because of overlapping binding sites, NF-I binding and Sp1 binding appear to be mutually exclusive. Overexpression of NF-I in cotransfection experiments with the alpha 1(I) promoter in NIH 3T3 fibroblasts increased alpha 1(I) expression, while Sp1 overexpression reduced this effect, as well as basal promoter activity. The herpes simplex virus thymidine kinase promoter, which contains independent NF-I- and Sp1-binding sites, was stimulated by both factors. Therefore, expression of the collagen alpha 1(I) gene may depend on the relative activities of NF-I and Sp1.

Enhanced expression of nuclear factor I/B in oxaliplatin-resistant human cancer cell lines

2010 ◽  
Vol 102 (2) ◽  
pp. 382-386 ◽  
Author(s):  
Eiji Kashiwagi ◽  
Hiroto Izumi ◽  
Yoshihiro Yasuniwa ◽  
Ryoko Baba ◽  
Yoshiaki Doi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Human Cancer ◽  
Cancer Cell Lines ◽  
Nuclear Factor ◽  
Human Cancer Cell ◽  
Human Cancer Cell Lines ◽  
Factor I ◽  
Enhanced Expression ◽  
Nuclear Factor I

Transcription factors nuclear factor I and Sp1 interact with the murine collagen alpha 1 (I) promoter

1991 ◽  
Vol 11 (8) ◽  
pp. 4065-4073
Author(s):  
M C Nehls ◽  
R A Rippe ◽  
L Veloz ◽  
D A Brenner
Keyword(s):  
Binding Sites ◽  
Direct Repeat ◽  
Dnase I ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Factor I ◽  
3T3 Fibroblasts ◽  
Nuclear Factor I ◽  
Simplex Virus ◽  
Nih 3T3

The collagen alpha 1(I) promoter, which is efficiently transcribed in NIH 3T3 fibroblasts, contains four binding sites for trans-acting factors, as demonstrated by DNase I protection assays (D. A. Brenner, R. A. Rippe, and L. Veloz, Nucleic Acids Res. 17:6055-6064, 1989). This study characterizes the DNA-binding proteins that interact with the two proximal footprinted regions, both of which contain a reverse CCAAT box and a G + C-rich 12-bp direct repeat. Analysis by DNase I protection assays, mobility shift assays, competition with specific oligonucleotides, binding with recombinant proteins, and reactions with specific antisera showed that the transcriptional factors nuclear factor I (NF-I) and Sp1 bind to these two footprinted regions. Because of overlapping binding sites, NF-I binding and Sp1 binding appear to be mutually exclusive. Overexpression of NF-I in cotransfection experiments with the alpha 1(I) promoter in NIH 3T3 fibroblasts increased alpha 1(I) expression, while Sp1 overexpression reduced this effect, as well as basal promoter activity. The herpes simplex virus thymidine kinase promoter, which contains independent NF-I- and Sp1-binding sites, was stimulated by both factors. Therefore, expression of the collagen alpha 1(I) gene may depend on the relative activities of NF-I and Sp1.

scholarly journals Purification of a cellular, double-stranded DNA-binding protein required for initiation of adenovirus DNA replication by using a rapid filter-binding assay.

1986 ◽  
Vol 6 (5) ◽  
pp. 1363-1373 ◽  
Author(s):  
J F Diffley ◽  
B Stillman
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Binding Assay ◽  
Binding Activity ◽  
Nuclear Factor ◽  
Factor I ◽  
Dna Binding Activity ◽  
Nuclear Extracts ◽  
Nuclear Factor I ◽  
Dna Fragment

A rapid and quantitative nitrocellulose filter-binding assay is described for the detection of nuclear factor I, a HeLa cell sequence-specific DNA-binding protein required for the initiation of adenovirus DNA replication. In this assay, the abundant nonspecific DNA-binding activity present in unfractionated HeLa nuclear extracts was greatly reduced by preincubation of these extracts with a homopolymeric competitor DNA. Subsequently, specific DNA-binding activity was detected as the preferential retention of a labeled 48-base-pair DNA fragment containing a functional nuclear factor I binding site compared with a control DNA fragment to which nuclear factor I did not bind specifically. This specific DNA-binding activity was shown to be both quantitative and time dependent. Furthermore, the conditions of this assay allowed footprinting of nuclear factor I in unfractionated HeLa nuclear extracts and quantitative detection of the protein during purification. Using unfrozen HeLa cells and reagents known to limit endogenous proteolysis, nuclear factor I was purified to near homogeneity from HeLa nuclear extracts by a combination of standard chromatography and specific DNA affinity chromatography. Over a 400-fold purification of nuclear factor I, on the basis of the specific activity of both sequence-specific DNA binding and complementation of adenovirus DNA replication in vitro, was affected by this purification. The most highly purified fraction was greatly enriched for a polypeptide of 160 kilodaltons on silver-stained sodium dodecyl sulfate-polyacrylamide gels. Furthermore, this protein cosedimented with specific DNA-binding activity on glycerol gradients. That this fraction indeed contained nuclear factor I was demonstrated by both DNase I footprinting and its function in the initiation of adenovirus DNA replication. Finally, the stoichiometry of specific DNA binding by nuclear factor I is shown to be most consistent with 2 mol of the 160-kilodalton polypeptide binding per mol of nuclear factor I-binding site.

scholarly journals Two Promoters Mediate Transcription from the Human LHX3 Gene: Involvement of Nuclear Factor I and Specificity Protein 1

Endocrinology ◽  
2006 ◽  
Vol 147 (1) ◽  
pp. 324-337 ◽  
Author(s):  
Benjamin C. Yaden ◽  
Marin Garcia ◽  
Timothy P. L. Smith ◽  
Simon J. Rhodes
Keyword(s):  
Human Gene ◽  
Nervous System Development ◽  
Hormone Deficiency ◽  
Comparative Approach ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Specificity Protein 1 ◽  
Factor I ◽  
Nuclear Factor I ◽  
Specificity Protein

The LHX3 transcription factor is required for pituitary and nervous system development in mammals. Mutations in the human gene are associated with hormone-deficiency diseases. The gene generates two mRNAs, hLHX3a and hLHX3b, which encode three proteins with different properties. Here, the cis elements and trans-acting factors that regulate the basal transcription of the two mRNAs are characterized. A comparative approach was taken featuring analysis of seven mammalian Lhx3 genes, with a focus on the human gene. Two conserved, TATA-less, GC-rich promoters that are used to transcribe the mRNAs precede exons 1a and 1b of hLHX3. Transcription start sites were mapped for both promoters. Deletion experiments showed most activity for reporter genes containing the basal promoters in the context of −2.0 kb of hLHX3a and 1.8 kb of intron 1a (hLHX3b). Transfection, site-directed mutation, electrophoretic mobility shift, Southwestern blot, and chromatin immunoprecipitation approaches were used to characterize the interaction of transcription factors with conserved elements in the promoters. Specificity protein 1 is a regulator of both promoters through interaction with GC boxes. In addition, a distal element within intron 1a that is recognized by nuclear factor I is critical for hLHX3b promoter function. We conclude that dual promoters allow regulated production of two hLHX3 mRNAs.

Purification of a cellular, double-stranded DNA-binding protein required for initiation of adenovirus DNA replication by using a rapid filter-binding assay

1986 ◽  
Vol 6 (5) ◽  
pp. 1363-1373
Author(s):  
J F Diffley ◽  
B Stillman
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Binding Assay ◽  
Binding Activity ◽  
Nuclear Factor ◽  
Factor I ◽  
Dna Binding Activity ◽  
Nuclear Extracts ◽  
Nuclear Factor I ◽  
Dna Fragment

A rapid and quantitative nitrocellulose filter-binding assay is described for the detection of nuclear factor I, a HeLa cell sequence-specific DNA-binding protein required for the initiation of adenovirus DNA replication. In this assay, the abundant nonspecific DNA-binding activity present in unfractionated HeLa nuclear extracts was greatly reduced by preincubation of these extracts with a homopolymeric competitor DNA. Subsequently, specific DNA-binding activity was detected as the preferential retention of a labeled 48-base-pair DNA fragment containing a functional nuclear factor I binding site compared with a control DNA fragment to which nuclear factor I did not bind specifically. This specific DNA-binding activity was shown to be both quantitative and time dependent. Furthermore, the conditions of this assay allowed footprinting of nuclear factor I in unfractionated HeLa nuclear extracts and quantitative detection of the protein during purification. Using unfrozen HeLa cells and reagents known to limit endogenous proteolysis, nuclear factor I was purified to near homogeneity from HeLa nuclear extracts by a combination of standard chromatography and specific DNA affinity chromatography. Over a 400-fold purification of nuclear factor I, on the basis of the specific activity of both sequence-specific DNA binding and complementation of adenovirus DNA replication in vitro, was affected by this purification. The most highly purified fraction was greatly enriched for a polypeptide of 160 kilodaltons on silver-stained sodium dodecyl sulfate-polyacrylamide gels. Furthermore, this protein cosedimented with specific DNA-binding activity on glycerol gradients. That this fraction indeed contained nuclear factor I was demonstrated by both DNase I footprinting and its function in the initiation of adenovirus DNA replication. Finally, the stoichiometry of specific DNA binding by nuclear factor I is shown to be most consistent with 2 mol of the 160-kilodalton polypeptide binding per mol of nuclear factor I-binding site.

scholarly journals Thioltransferase (Glutaredoxin) Reactivates the DNA-binding Activity of Oxidation-inactivated Nuclear Factor I

1998 ◽  
Vol 273 (1) ◽  
pp. 392-397 ◽  
Author(s):  
Smarajit Bandyopadhyay ◽  
David W. Starke ◽  
John J. Mieyal ◽  
Richard M. Gronostajski
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Nuclear Factor ◽  
Factor I ◽  
Dna Binding Activity ◽  
Nuclear Factor I
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