scholarly journals A 39-kD DNA-binding protein from mouse brain stimulates transcription of myelin basic protein gene in oligodendrocytic cells.

1995 ◽  
Vol 130 (5) ◽  
pp. 1171-1179 ◽  
Author(s):  
S Haas ◽  
P Thatikunta ◽  
A Steplewski ◽  
E M Johnson ◽  
K Khalili ◽  
...  
Keyword(s):  
Dna Binding ◽  
Myelin Basic Protein ◽  
Mouse Brain ◽  
Basic Protein ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Regulatory Sequence ◽  
Major Protein ◽  
Cell Type ◽  
Cell Type Specific

The MB1 regulatory sequence of the myelin basic protein (MBP) gene spanning between nucleotides -14 to -50 with respect to the transcription start site is critical for cell type-specific transcription of the MBP gene, which encodes the major protein component of myelin sheath in cells derived from the central nervous system (CNS). This regulatory sequence has the ability to interact with a developmentally controlled DNA-binding protein from mouse brain that stimulates transcription of MBP promoter in an in vitro system (Haas, S., J. Gordon, and K. Khalili. 1993. Mol. Cell. Biol. 13:3103-3112). Here, we report the purification of a 39-kD protein from mouse brain tissue at the peak of myelination and MBP production that binds to the MB1 regulatory motif. Following partial amino acid sequence analysis, we have identified a complementary DNA encoding a 39-kD DNA-binding protein called pur alpha. Expression of pur alpha cDNA in the prokaryotic and eukaryotic cells resulted in the synthesis of a protein with characteristics similar to the purified brain-derived 39-kD protein in band shift competition assays. Cotransfection of the recombinant pur alpha expressor plasmid with MBP promoter construct indicated that Pur alpha stimulates transcription of the MBP promoter in oligodendrocytic cells, and that the nucleotide sequence required for binding of the 39-kD Pur alpha to DNA within the MB1 region is crucial for this activity. Moreover, transient expression of Pur alpha caused elevation in the level of endogenous MBP RNA in oligodendrocytic cells. Thus, Pur alpha, a sequence-specific DNA-binding protein upon binding to MB1 regulatory region may play a significant role in determining the cell type-specific expression of MBP in brain.

A developmentally regulated DNA-binding protein from mouse brain stimulates myelin basic protein gene expression

1993 ◽  
Vol 13 (5) ◽  
pp. 3103-3112
Author(s):  
S Haas ◽  
J Gordon ◽  
K Khalili
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Dna Binding ◽  
Myelin Basic Protein ◽  
Mouse Brain ◽  
Basic Protein ◽  
Binding Protein ◽  
Cell Type ◽  
A Cell ◽  
Cell Type Specific

Transcription of the myelin basic protein (MBP) gene is regulated in a cell-type-specific and developmental stage-specific manner during myelin formation in the murine central nervous system. The 5'-flanking region of the MBP gene contains several regulatory elements that differentially contribute to the cell-type-specific transcription of MBP in cells derived from the central nervous system. The proximal element, termed MB1, which is located between nucleotides -14 and -50 with respect to the RNA start site, has previously been shown to have characteristics of a cell-type-specific enhancer element. In this study, we used band shift and UV cross-linking assays to identify DNA-binding proteins in mouse brain nuclear extract which interact with the MB1 element. Fractionation of these extracts has allowed the identification of a 38- to 41-kDa nuclear protein, derived from mouse brain tissue at the peak of myelination, which specifically binds the MB1 DNA sequence. Fractions enriched in the MB1-binding protein have been shown to stimulate transcription of the MBP promoter in extract derived from HeLa cells. MB1 binding protein activity is expressed in a tissue-specific and development stage-specific pattern which coincides with the pattern of MBP transcription, suggesting that this protein may be a biologically relevant transcription factor for the MBP gene in vivo.

scholarly journals A developmentally regulated DNA-binding protein from mouse brain stimulates myelin basic protein gene expression.

1993 ◽  
Vol 13 (5) ◽  
pp. 3103-3112 ◽  
Author(s):  
S Haas ◽  
J Gordon ◽  
K Khalili
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Dna Binding ◽  
Myelin Basic Protein ◽  
Mouse Brain ◽  
Basic Protein ◽  
Binding Protein ◽  
Cell Type ◽  
A Cell ◽  
Cell Type Specific

Transcription of the myelin basic protein (MBP) gene is regulated in a cell-type-specific and developmental stage-specific manner during myelin formation in the murine central nervous system. The 5'-flanking region of the MBP gene contains several regulatory elements that differentially contribute to the cell-type-specific transcription of MBP in cells derived from the central nervous system. The proximal element, termed MB1, which is located between nucleotides -14 and -50 with respect to the RNA start site, has previously been shown to have characteristics of a cell-type-specific enhancer element. In this study, we used band shift and UV cross-linking assays to identify DNA-binding proteins in mouse brain nuclear extract which interact with the MB1 element. Fractionation of these extracts has allowed the identification of a 38- to 41-kDa nuclear protein, derived from mouse brain tissue at the peak of myelination, which specifically binds the MB1 DNA sequence. Fractions enriched in the MB1-binding protein have been shown to stimulate transcription of the MBP promoter in extract derived from HeLa cells. MB1 binding protein activity is expressed in a tissue-specific and development stage-specific pattern which coincides with the pattern of MBP transcription, suggesting that this protein may be a biologically relevant transcription factor for the MBP gene in vivo.

scholarly journals a/Alpha-specific repression by MAT alpha 2.

Genetics ◽  
1988 ◽  
Vol 120 (1) ◽  
pp. 75-81
Author(s):  
J Strathern ◽  
B Shafer ◽  
J Hicks ◽  
C McGill
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Alpha Cell ◽  
Alpha Cells ◽  
Cell Type ◽  
New Class ◽  
Cell Type Specific

Abstract The product of the MAT alpha 2 gene is a DNA-binding protein that acts as a repressor of two different sets of cell type-specific genes. In alpha cells, the alpha 2 protein represses the transcription of several a-specific genes. In a/alpha cells, the alpha 2 protein acts together with the product of the MATa1 gene, the a1 protein, to repress several genes used by haploids in the mating process. In addition to the mat alpha 2 mutations that result in defects in both types of regulation, other mat alpha 2 alleles have been described that result in defects in the repression of a-specific genes but that do not affect the ability of the alpha 2 and a1 proteins to interact to repress the haploid-specific genes. We report here the isolation of a new class of mat alpha 2 mutations that do not affect the ability of the alpha 2 protein to repress a-specific genes, but that interfere with the ability of the alpha 2 protein to interact with the a1 protein to repress the haploid-specific genes and establish the a/alpha cell type. These mutations may help determine the means by which the a1 protein interacts with alpha 2 to expand the set of genes under its control.

Cell-type-specific transcriptional repression of H+, K+-ATPase a-subunit gene mediated by a 35-kDa nuclear DNA-binding protein

2000 ◽  
Vol 118 (4) ◽  
pp. A293
Author(s):  
Akira Muraoka ◽  
Mitsuru Kaise ◽  
Junko Yamada ◽  
Kei Matsueda ◽  
Ryosuke Shoda ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcriptional Repression ◽  
Nuclear Dna ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Subunit Gene ◽  
Cell Type ◽  
A Subunit ◽  
Cell Type Specific

scholarly journals Biochemical characterization of mouse brain necdin

1996 ◽  
Vol 314 (3) ◽  
pp. 895-901 ◽  
Author(s):  
Etsuko MARUYAMA
Keyword(s):  
Dna Binding ◽  
Affinity Chromatography ◽  
Mouse Brain ◽  
Silver Staining ◽  
Biochemical Characterization ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Micrococcal Nuclease ◽  
Dependent Manner ◽  
Chromatography Column

Necdin is a protein encoded by neural differentiation-specific mRNA derived from embryonal carcinoma cells (P19). Necdin of mouse brain was characterized by Western blotting and silver-staining analysis by using affinity purified antibodies to 17 synthetic peptides of deduced C-terminal amino acids. Necdin exhibits a molecular mass of 51 kDa on SDS/PAGE, and is localized in the S1 and S2 nucleosomal fractions. Sonicated necdin is found in all fractions of Sephacryl S-300 gel filtration chromatography, with a peak at 700 kDa. Necdin is released on micrococcal nuclease digestion, which is essential for electrophoretic migration on acetic acid/urea/Triton gels, suggesting that it could be a DNA-binding protein. Nucleosomal necdin shows two peaks at approx. 10 S and approx. 20 S on sucrose gradient centrifugation in the presence of 0.6 M NaCl, and a single peak in the presence of 2.0 M NaCl. Necdin forms a huge complex through chemical cross-linking with glutaraldehyde or dimethyl sulphate. The silver-staining intensity of the 51 kDa band corresponds to the decrease in the immuno-staining in a reagent concentration-dependent manner. Necdin binds tightly to a double-stranded DNA affinity chromatography column, and can be eluted from it with 2.0 M NaCl after washing with 0.6 M NaCl (approx. 100 ng per ml of gel). This purified necdin exhibits a pI of 9.1 on isoelectric focusing. The nucleosomal necdin complex (> 200 kDa) was adsorbed on an organomercurial agarose affinity chromatography column and was eluted with 10 mM DTT, revealing that necdin is possibly involved in the transactive nucleosomal complex. These data show that necdin is a nuclear basic DNA-binding protein that associates with other molecules to regulate transcriptionally active genes and nuclear function.

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