Abstract
We have identified a novel transcription unit of 105 kilobases (called the Golli-mbp gene) that encompasses the mouse myelin basic protein (MBP) gene. Three unique exons within this gene are alternatively spliced into MBP exons and introns to produce a family of MBP gene-related mRNAs that are under individual developmental regulation. These mRNAs are temporally expressed within cells of the oligodendrocyte lineage at progressive stages of differentiation. Thus, the MBP gene is a part of a more complex gene structure, the products of which may play a role in oligodendrocyte differentiation prior to myelination. One Golli-mbp mRNA that encodes a protein antigenically related to MBP is also expressed in the spleen and other non-neural tissues.
Highlights
In the central nervous system, myelination is a developmentally regulated event that requires the precise coordination of expression of a number of myelin proteins and lipids (Campagnoni and Macklin, 1988).During late embryonic and neonatal murine brain development, oligodendrocyte precursor cells differentiate intoimmature oligodendrocytes (see Cameron and Rakic (1991) for review)
The alternatively spliced myelin basic protein (MBP) mRNAs encode MBP isoforms that range in molecular mass from14 to 21 kDa
We reported the existence of M41-MBP mRNA, which we believe is produced from a transcription start site upstream of the classic MBP gene transcription start site (Kitamura et al, 1990)
Summary
Northern Blots and RNA Isolation-RNAwas isolated by the guanidinium thiocyanate method and purified through acesium chloride gradient (MacDonald et al, 1987). Puked-field Gel Electrophoresis-Mouse lymphocytes were prepared and resuspended in phosphate-buffered saline at a concentration of 5 X lo' cells/ml. Combined in Situ Hybridization/Zmmunocytochemistry of Cell Cultures-Primary cultures were prepared as described by Amur-Umarjee et al (1990a), and enriched oligodendrocyteswere purified by the method of Suzumura et al (1984). DNA probes were labeled with digoxigenin as previously described (Amur-Umarjee et aZ., 1990b).A combination of nonradioactive in situ hybridization and immunofluorescence techniques (Amur-Umajee et al, 1990a) was employed to localize Golli-mbpgene transcripts with markers of cells in the oligodendroglial lineage. A confocal imaging system (Bio-Rad MRC-500) was utilized to enhance the fluorescence images and to reverse the phase contrast of the cells labeledby in situ hybridization Antibodies used in these studies were generously provided by Dr Kari Stefansson (A007) and Dr Joyce Benjamins (A2B5 and antigalactocerebroside). Digoxigenin-labeled riboprobes were prepared using digoxigenin-labeled UTP as described by the manufacturer (Boehringer Mannheim) and transcribed from either the T7or T3 RNA polymerase promoter to produce sense or antisense riboprobes
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