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Nuclear proteins in mouse brain cells bind specifically to the myelin basic protein regulatory region.

ABSTRACT: Expression of myelin basic protein (MBP) in mice is regulated in a cell- and stage-specific manner during brain development. The MBP control region contains multiple cis-acting elements, shown by in vivo and in vitro assays, which are responsible for its unique pattern of transcription. Using synthetic DNA fragments spanning the MBP control region, we have analyzed nuclear proteins obtained from newborn (2-3 d), young adult (18-30 d), and adult (60 d) animals; these nuclear proteins form DNA-protein complexes with the MBP regulatory region. Brain extracts from young adult and adult mice showed enhanced binding activities with the sequences supporting transcriptional activation in glial cells. Deletion analysis of the proximal activating sequence located at position -14 to -50 with respect to the RNA initiation site resulted in identification of a small region, located between nucleotides -14 to -37, which is required for formation of the complexes. Southwestern assay revealed a major 39-kD protein from young adult brain extract that recognizes the sequences between nucleotides -14 to -37. An additional minor 37-kD protein, derived from young adult brain extract, was also found to be associated with this proximal activating region. Of particular interest is the observation that the minor 37-kD protein became more abundant in the extract derived from adult brain, whereas the major 39-kD protein became less abundant. The possible role of these proteins in cell/stage-specific transcription of MBP is discussed.

SUBMITTER: Lashgari MS

PROVIDER: S-EPMC296918 | biostudies-other | 1990 Nov

REPOSITORIES: biostudies-other

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Nuclear proteins in mouse brain cells bind specifically to the myelin basic protein regulatory region.

Lashgari M S MS Devine-Beach K K Haas S S Khalili K K

The Journal of clinical investigation 19901101 5

Expression of myelin basic protein (MBP) in mice is regulated in a cell- and stage-specific manner during brain development. The MBP control region contains multiple cis-acting elements, shown by in vivo and in vitro assays, which are responsible for its unique pattern of transcription. Using synthetic DNA fragments spanning the MBP control region, we have analyzed nuclear proteins obtained from newborn (2-3 d), young adult (18-30 d), and adult (60 d) animals; these nuclear proteins form DNA-pro ...[more]

PMID: 1700799

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Project description:When rat brain myelin was examined by sodium dodecyl sulphate/polyacrylamideslab-gel electrophoresis followed by fluorography of the stained gel, it was found that a host of proteins of rat brain myelin were labelled 2, 4 and 24h after the intracerebral injection of H(3) (32)PO(4). Among those labelled were proteins migrating to the positions of myelin-associated glycoprotein, Wolfgram proteins, proteolipid protein, DM-20 and basic proteins. The four basic proteins with mol.wts. 21000, 18000 (large basic protein), 17000 and 14000 (small basic protein) were shown to be phosphorylated after electrophoresis in both acid-urea- and sodium dodecyl sulphate-containing gel systems followed by fluorography. The four basic proteins imparted bluish-green colour, after staining with Amido Black, which is characteristic of myelin basic proteins. The four basic proteins were purified to homogeneity. Fluorography of the purified basic proteins after re-electrophoresis revealed the presence of phosphorylated high-molecular-weight ;polymers' associated with each basic protein. The amino acid compositions of the phosphorylated large basic protein and small basic proteins are compatible with the amino acid sequences. Proteins with mol.wts. 21000 and 17000 gave the expected amino acid composition of myelin basic proteins. Radiolabelled phosphoserine and phosphothreonine were identified after partial acid hydrolysis of the four purified basic proteins. The [(32)P]phosphate-protein bond in the basic protein was stable at an acidic pH but was readily hydrolysed at alkaline pH, as would be expected of phosphoester bonds involving both serine and threonine residues. Double-immunodiffusion analysis demonstrated that the four phosphorylated proteins showed complete homology when diffused against antiserum to a mixture of small and large basic proteins. Since the four basic proteins of rat brain myelin were phosphorylated both in vivo and in vitro it is postulated that the same protein kinase is responsible for their phosphorylation in both conditions.

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Nuclear proteins in mouse brain cells bind specifically to the myelin basic protein regulatory region.

Publications

Nuclear proteins in mouse brain cells bind specifically to the myelin basic protein regulatory region.

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