STRUCTURAL ORGANIZATION OF MYELIN PROTEIN GENES

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In this application, we propose to test two new hypotheses concerning the MBP gene. These hypotheses are based upon data derived from the identification of two classes of unusual myelin basic protein (MBP)-related transcripts isolated from screens of mouse brain and human fetal spinal cord cDNA libraries. The first hypothesis is that the structure of the MBP gene is presently incomplete and contains at least one more exon upstream of what is currently believed to be exon 1 and an additional promoter. The second hypothesis is that portions of the MBP gene (including parts of at least one exon and one intron) are transcribed into mRNAs encoding proteins other than the MBP variants characterized thus far. The location of any additional exon sequences will be determined by conventional procedures of mapping and sequencing genomic clones using an MBP cDNA that contains sequence corresponding to the additional exon region. Sites of transcription initiation at this new upstream exon will be determined by S-1 nuclease and primer extension analysis. Experiments will be performed to determine if initiation of transcription at this start site and the one previously identified in the MBP gene varies during development or in different regions of the CNS. Experiments will also be performed to determine if all MBP variant forms are produced when transcription begins at this new transcription site. Full length cDNAs corresponding to the second class of MBP-gene related transcripts will be isolated, sequenced, and characterized. The structural relationship between this second class of transcripts and the MBP gene will be determined. The intron/exon arrangement of the gene coding for these MBP-related cDNAs will be elucidated and it will be determined if all the exons of this gene are encoded by genomic segments completely included within the MBP gene or if some exons lie outside the MBP gene. To determine if these transcripts are expressed in tissues other than brain or in cell types other than oligodendrocytes, Northern blot analyses of mRNA from a variety of tissues and cultured cells will be performed. The developmental appearance of the transcripts in the brains of normal mice and dysmyelinating mutants will be compared with the expression of the MBP mRNAs. In situ hybridization studies will be performed to determine the cellular and regional localization of the transcripts.