scholarly journals Rapid detection and purification of sequence specific DNA binding proteins using magnetic separation

2006 ◽  
Vol 71 (2) ◽  
pp. 135-141 ◽  
Author(s):  
Marija Mojsin ◽  
Jelena Djurovic ◽  
Isidora Petrovic ◽  
Aleksandar Krstic ◽  
Danijela Drakulic ◽  
...  
Keyword(s):  
Dna Binding ◽  
Magnetic Separation ◽  
Magnetic Particles ◽  
Binding Proteins ◽  
Rapid Identification ◽  
Dna Binding Proteins ◽  
Binding Activity ◽  
High Yield ◽  
Dna Binding Activity ◽  
Putative Binding Site

In this paper, a method for the rapid identification and purification of sequence specific DNA binding proteins based on magnetic separation is presented. This method was applied to confirm the binding of the human recombinant USF1 protein to its putative binding site (E-box) within the human SOX3 protomer. It has been shown that biotinylated DNA attached to streptavidin magnetic particles specifically binds the USF1 protein in the presence of competitor DNA. It has also been demonstrated that the protein could be successfully eluted from the beads, in high yield and with restored DNA binding activity. The advantage of these procedures is that they could be applied for the identification and purification of any high-affinity sequence-specific DNA binding protein with only minor modifications.

Interaction of two sequence-specific single-stranded DNA-binding proteins with an essential region of the beta-casein gene promoter is regulated by lactogenic hormones

1993 ◽  
Vol 13 (12) ◽  
pp. 7303-7310
Author(s):  
S Altiok ◽  
B Groner
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Mammary Epithelial Cells ◽  
Dna Binding Proteins ◽  
Binding Activity ◽  
Mammary Epithelial ◽  
Nuclear Proteins ◽  
Single Stranded Dna ◽  
Dna Binding Activity ◽  
Beta Casein

Transcription of the beta-casein gene in mammary epithelial cells is regulated by the lactogenic hormones insulin, glucocorticoids, and prolactin. The DNA sequence elements in the promoter which confer the action of the hormones on the transcriptional machinery and the nuclear proteins binding to this region have been investigated. We found that 221 nucleotides of promoter sequence 5' of the RNA start site are sufficient to mediate the induction of a chloramphenicol acetyltransferase reporter gene in transfected HC11 mammary epithelial cells. Deletion of 5' sequences to position -183 results in a construct with enhanced basal activity which still retains inducibility. A -170 beta-casein promoter-chloramphenicol acetyltransferase construct has very low transcriptional activity, which indicates the presence of a negative regulatory in the region between -221 and -183 and a positive regulatory element between -183 and -170. Band shift analysis showed that the promoter region between -194 and -163 specifically binds two nuclear proteins. The proteins are sequence-specific, single-stranded DNA-binding proteins which exclusively recognize the upper DNA strand and most likely play a repressing role in transcription. DNA binding activity of these nuclear proteins was observed only in nuclear extracts from mammary glands of mice in late pregnancy and postlactation, not during lactation. Hormonal control of the DNA binding activity of these proteins was also observed in the mammary epithelial cell line HC11. Mixing experiments showed that extracts from mammary tissue of lactating mice and from lactogenic hormone-treated HC11 cells contain an activity which can suppress the DNA binding of the single-stranded DNA-binding proteins.2+ identical specificity to the single-stranded DNA.

scholarly journals Interaction of two sequence-specific single-stranded DNA-binding proteins with an essential region of the beta-casein gene promoter is regulated by lactogenic hormones.

1993 ◽  
Vol 13 (12) ◽  
pp. 7303-7310 ◽  
Author(s):  
S Altiok ◽  
B Groner
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Mammary Epithelial Cells ◽  
Dna Binding Proteins ◽  
Binding Activity ◽  
Mammary Epithelial ◽  
Nuclear Proteins ◽  
Single Stranded Dna ◽  
Dna Binding Activity ◽  
Beta Casein

Transcription of the beta-casein gene in mammary epithelial cells is regulated by the lactogenic hormones insulin, glucocorticoids, and prolactin. The DNA sequence elements in the promoter which confer the action of the hormones on the transcriptional machinery and the nuclear proteins binding to this region have been investigated. We found that 221 nucleotides of promoter sequence 5' of the RNA start site are sufficient to mediate the induction of a chloramphenicol acetyltransferase reporter gene in transfected HC11 mammary epithelial cells. Deletion of 5' sequences to position -183 results in a construct with enhanced basal activity which still retains inducibility. A -170 beta-casein promoter-chloramphenicol acetyltransferase construct has very low transcriptional activity, which indicates the presence of a negative regulatory in the region between -221 and -183 and a positive regulatory element between -183 and -170. Band shift analysis showed that the promoter region between -194 and -163 specifically binds two nuclear proteins. The proteins are sequence-specific, single-stranded DNA-binding proteins which exclusively recognize the upper DNA strand and most likely play a repressing role in transcription. DNA binding activity of these nuclear proteins was observed only in nuclear extracts from mammary glands of mice in late pregnancy and postlactation, not during lactation. Hormonal control of the DNA binding activity of these proteins was also observed in the mammary epithelial cell line HC11. Mixing experiments showed that extracts from mammary tissue of lactating mice and from lactogenic hormone-treated HC11 cells contain an activity which can suppress the DNA binding of the single-stranded DNA-binding proteins.2+ identical specificity to the single-stranded DNA.

Degradation of constitutive transcription factors during apoptosis in rat thymocytes

1994 ◽  
Vol 72 (11-12) ◽  
pp. 639-648 ◽  
Author(s):  
Ian de Belle ◽  
Lucia Testolin ◽  
Siyaram Pandey ◽  
Christine Carson ◽  
P. Roy Walker ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Dna Binding ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Binding Activity ◽  
Significant Loss ◽  
Dna Binding Activity ◽  
The Stability ◽  
Induced Apoptosis

Dexamethasone (Dex) accelerates the rate of apoptosis in thymocytes by a process thought to require gene expression. Among the genes implicated in the regulation of this phenomenon are the immediate early genes such as c-fos and c-jun, whose expression is modulated by a complement of preexisting transcription factors. We have analyzed the DNA-binding activity of these constitutive transcription factors during Dex-induced apoptosis in thymocytes to assess their functionality. We observed a progressive loss of the DNA-binding proteins in parallel with the appearance of the characteristic morphological and biochemical features of apoptosis. At the same time we have found a general increase in the nuclear proteolytic activity concomitant with a significant loss of the nuclear nonhistone chromosomal proteins. Indeed, cotreatment of thymocytes with the nonspecific serine protease inhibitor phenyl-methylsulphonyl fluoride was able to partially protect the stability of the DNA-binding proteins and alter the expression of the c-fos and c-jun genes but did not inhibit apoptosis. Our results suggest that the action of a protease(s) is responsible for the degradation of constitutive transcription factors during Dex-induced apoptosis, rendering the death pathway irreversible.Key words: apoptosis, thymocytes, proteolysis, transcription factors, gene expression.

Analysis of epidermal proteins for DNA-binding activity

1987 ◽  
Vol 7 (8) ◽  
pp. 645-651
Author(s):  
Prakash C. Supakar ◽  
Renuka Misra ◽  
Gopal M. Bhatnagar
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Binding Activity ◽  
Soluble Extract ◽  
Newborn Rat ◽  
Molecular Weights ◽  
Dna Binding Activity ◽  
Cellulose Column

A group of DNA-binding proteins from the soluble extract of newborn rat epidermis have been separated by chromatography using DNA-cellulose columns. The electrophoretogram of the DNA-binding proteins eluted from a single stranded DNA-cellulose column shows five major proteins of molecular weights ranging between 25K to 40K. Both the epidermal protein filaggrin and most keratins, except two high molecular weight keratins, do not show in vitro DNA-binding activity.

Identification of single-stranded-DNA-binding proteins that interact with muscle gene elements

1991 ◽  
Vol 11 (4) ◽  
pp. 1944-1953
Author(s):  
I M Santoro ◽  
T M Yi ◽  
K Walsh
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Regulatory Element ◽  
Dna Binding Proteins ◽  
Binding Activity ◽  
Sequence Motif ◽  
Mobility Shift ◽  
Specific Sequence ◽  
Single Stranded Dna ◽  
Muscle Gene

A sequence-specific DNA-binding protein from skeletal-muscle extracts that binds to probes of three muscle gene DNA elements is identified. This protein, referred to as muscle factor 3, forms the predominant nucleoprotein complex with the MCAT gene sequence motif in an electrophoretic mobility shift assay. This protein also binds to the skeletal actin muscle regulatory element, which contains the conserved CArG motif, and to a creatine kinase enhancer probe, which contains the E-box motif, a MyoD-binding site. Muscle factor 3 has a potent sequence-specific, single-stranded-DNA-binding activity. The specificity of this interaction was demonstrated by sequence-specific competition and by mutations that diminished or eliminated detectable complex formation. MyoD, a myogenic determination factor that is distinct from muscle factor 3, also bound to single-stranded-DNA probes in a sequence-specific manner, but other transcription factors did not. Multiple copies of the MCAT motif activated the expression of a heterologous promoter, and a mutation that eliminated expression was correlated with diminished factor binding. Muscle factor 3 and MyoD may be members of a class of DNA-binding proteins that modulate gene expression by their abilities to recognize DNA with unusual secondary structure in addition to specific sequence.

scholarly journals Identification of single-stranded-DNA-binding proteins that interact with muscle gene elements.

1991 ◽  
Vol 11 (4) ◽  
pp. 1944-1953 ◽  
Author(s):  
I M Santoro ◽  
T M Yi ◽  
K Walsh
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Regulatory Element ◽  
Dna Binding Proteins ◽  
Binding Activity ◽  
Sequence Motif ◽  
Mobility Shift ◽  
Specific Sequence ◽  
Single Stranded Dna ◽  
Muscle Gene

A sequence-specific DNA-binding protein from skeletal-muscle extracts that binds to probes of three muscle gene DNA elements is identified. This protein, referred to as muscle factor 3, forms the predominant nucleoprotein complex with the MCAT gene sequence motif in an electrophoretic mobility shift assay. This protein also binds to the skeletal actin muscle regulatory element, which contains the conserved CArG motif, and to a creatine kinase enhancer probe, which contains the E-box motif, a MyoD-binding site. Muscle factor 3 has a potent sequence-specific, single-stranded-DNA-binding activity. The specificity of this interaction was demonstrated by sequence-specific competition and by mutations that diminished or eliminated detectable complex formation. MyoD, a myogenic determination factor that is distinct from muscle factor 3, also bound to single-stranded-DNA probes in a sequence-specific manner, but other transcription factors did not. Multiple copies of the MCAT motif activated the expression of a heterologous promoter, and a mutation that eliminated expression was correlated with diminished factor binding. Muscle factor 3 and MyoD may be members of a class of DNA-binding proteins that modulate gene expression by their abilities to recognize DNA with unusual secondary structure in addition to specific sequence.

scholarly journals Dependence on androgens of the specific DNA-binding activity of rat ventral-prostate non-histone chromosomal proteins

1981 ◽  
Vol 194 (1) ◽  
pp. 91-98
Author(s):  
B H Lesser ◽  
N L Elliot
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Specific Binding ◽  
Binding Activity ◽  
Male Rats ◽  
Ventral Prostate ◽  
Trace Quantity ◽  
Chromosomal Proteins ◽  
Dna Binding Activity ◽  
Rat Prostate

Interactions between rat prostate non-histone chromosomal proteins and DNA were studied by using a nitrocellulose-filter-binding technique to monitor the formation of DNA–protein complexes. The total binding activity of the non-histones, as measured by binding of proteins to a trace quantity of labelled DNA, displays no preference for rat DNA relative to Escherichia coli DNA. Sequestration of non-specific binding proteins by preincubation with unlabelled bacterial DNA enables detection of a fraction of rat prostate non-histones that binds preferentially to labelled rat DNA relative to labelled E. coli DNA. After castration of adult male rats, both total and specific binding activities decrease. Administration of 5 alpha-dihydrotestosterone to castrated rats stimulates both total and specific DNA-binding activities of prostate non-histones; specific binding is stimulated to a greater extent than total DNA, indicating that the specific binding proteins constitute a larger fraction of the non-histone proteins in the presence of androgens. The specific DNa-binding activity is dependent on the dose of steroid administered.

scholarly journals Increased expression and DNA-binding activity of transcription factor Sp1 in doxorubicin-resistant HL-60 leukemia cells.

1990 ◽  
Vol 10 (10) ◽  
pp. 5541-5547 ◽  
Author(s):  
F Borellini ◽  
A Aquino ◽  
S F Josephs ◽  
R I Glazer
Keyword(s):  
Human Immunodeficiency Virus ◽  
Dna Binding ◽  
Dna Binding Proteins ◽  
Binding Activity ◽  
Leukemia Cells ◽  
Nf Kappa B ◽  
Dna Binding Activity ◽  
Nuclear Extracts ◽  
Immunodeficiency Virus

The processes responsible for the multidrug-resistant (Mdr) phenotype in Adriamycin (doxorubicin)-resistant HL-60 leukemia cells (HL-60/AR) are not defined. Since enhanced transcription of resistance-related proteins is associated with Mdr cells, we sought to determine whether changes in the expression of specific transcription factors were a feature characteristic of the Mdr process. Nuclear extracts were prepared from wild-type and resistant cells and compared for their ability to bind DNA consensus sequences for the transcription factors Sp1 and NF kappa B contained in the 5' long terminal repeat region of human immunodeficiency virus type 1. Southwestern (DNA-protein) blots showed a family of DNA-binding proteins of 105 kilodaltons (kDa) that were present only in HL-60/AR cells. Competitive gel shift assays indicated that these factors were related to transcription factor Sp1, and immunoblotting with an Sp1 antibody identified this factor as Sp1. DNase footprinting of the promoter region in the human immunodeficiency virus type 1 5' long terminal repeat showed that protection occurred at two Sp1 sites as well as two NF kappa B sites and the trans-acting region with nuclear extracts only from resistant cells. Preliminary evidence also suggests that phosphorylation may play a negative regulatory role in the activity of Sp1, since calf intestine alkaline phosphatase stimulated the DNA-binding activity of Sp1 in vitro. These results indicate that HL-60/AR cells contain an abundance of DNA-binding proteins, particularly Sp1, which probably interact with other cis-acting regulatory proteins in a cooperative manner.

Increased expression and DNA-binding activity of transcription factor Sp1 in doxorubicin-resistant HL-60 leukemia cells

1990 ◽  
Vol 10 (10) ◽  
pp. 5541-5547
Author(s):  
F Borellini ◽  
A Aquino ◽  
S F Josephs ◽  
R I Glazer
Keyword(s):  
Human Immunodeficiency Virus ◽  
Dna Binding ◽  
Dna Binding Proteins ◽  
Binding Activity ◽  
Leukemia Cells ◽  
Nf Kappa B ◽  
Dna Binding Activity ◽  
Nuclear Extracts ◽  
Immunodeficiency Virus

The processes responsible for the multidrug-resistant (Mdr) phenotype in Adriamycin (doxorubicin)-resistant HL-60 leukemia cells (HL-60/AR) are not defined. Since enhanced transcription of resistance-related proteins is associated with Mdr cells, we sought to determine whether changes in the expression of specific transcription factors were a feature characteristic of the Mdr process. Nuclear extracts were prepared from wild-type and resistant cells and compared for their ability to bind DNA consensus sequences for the transcription factors Sp1 and NF kappa B contained in the 5' long terminal repeat region of human immunodeficiency virus type 1. Southwestern (DNA-protein) blots showed a family of DNA-binding proteins of 105 kilodaltons (kDa) that were present only in HL-60/AR cells. Competitive gel shift assays indicated that these factors were related to transcription factor Sp1, and immunoblotting with an Sp1 antibody identified this factor as Sp1. DNase footprinting of the promoter region in the human immunodeficiency virus type 1 5' long terminal repeat showed that protection occurred at two Sp1 sites as well as two NF kappa B sites and the trans-acting region with nuclear extracts only from resistant cells. Preliminary evidence also suggests that phosphorylation may play a negative regulatory role in the activity of Sp1, since calf intestine alkaline phosphatase stimulated the DNA-binding activity of Sp1 in vitro. These results indicate that HL-60/AR cells contain an abundance of DNA-binding proteins, particularly Sp1, which probably interact with other cis-acting regulatory proteins in a cooperative manner.

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