We present the complete nucleotide sequence of a cDNA encoding rat cyclophilin. The 743-nucleotide sequence contains a 42-nucleotide 5' noncoding region, a 492 nucleotide open reading frame corresponding to a translation product of 164 amino acids with a molecular weight of 17,874, and a 3' noncoding region of 209 nucleotides. Primer extension studies reveal the presence of one minor and two major transcription start sites. Southern blot analyses are consistent with as many as 20 copies of the cyclophilin gene and possible pseudogenes. Cyclophilin mRNA is expressed in virtually all types of tissues of rat and monkey and appears to have been highly conserved during mammalian evolution.

No abstract

Proteolipid protein (PLP) is the major myelin membrane protein of the central nervous system. We have isolated a copy of an alternatively spliced PLP gene transcript from a mouse brain cDNA library that was screened for PLP-related sequences. The encoded 241-amino acid protein differs from PLP by an internal deletion of 35-amino acid residues (116-150) from the major hydrophilic domain. This PLP variant is identical with the DM-20 protein of myelin, previously described as a brain-specific myelin component and known to be related to PLP. We determined the corresponding nucleotide sequence of the rat PLP gene and found that DM-20 mRNA results when a second 5' splice site, located 105 nucleotides within the third exon of the primary PLP transcript, is utilized in precursor mRNA (pre-mRNA) splicing. This demonstrates that alternative 5' splice site selection can determine the protein product of a cellular gene. DM-20 mRNA is expressed in rat brain with approximately 50% abundance relative to PLP mRNA and appears to be developmentally coregulated. Myelination in the mammalian central nervous system (CNS)is the specialized function of differentiated oligodendrocytes and involves the coordinated expression of myelin-specific gene products. Proteolipid protein (PLP) is the most abundant myelin protein of the CNS and is a highly hydrophobic integral membrane protein (reviewed in ref. 1). Its primary structure has been established by protein sequencing (2-4) and more recently from the cloning of PLP cDNAs from several species (5-10). A mutation in the mouse PLP gene is the primary genetic defect of the dysmyelinating mutant mouse jimpy (8-12), which demonstrates the critical role of PLP in CNS myelin assembly.Another CNS specific myelin protein of lower abundance has been termed "intermediate protein" or DM-20 (13), which reflects its apparent molecular mass. Depending on the proteolipid extraction method (14), DM-20 copurifies with PLP, but the two proteins can easily be separated by NaDodSO4 gel electrophoresis. PLP and the DM-20 protein are related by amino-and carboxyl-terminal amino acid sequence homology and immunological crossreactivity (1). The exact structure of DM-20 and its relationship to PLP had been controversial, but recent evidence has suggested that DM-20 differs from PLP by an internal deletion of residues 100-140 (plus or minus a few amino acids) (15, 16). To determine the exact relationship between PLP and DM-20 and to test the possibility that the DM-20 protein is a result of alternative PLP precursor mRNA (pre-mRNA) splicing, we screened a mouse brain cDNA library for PLP-related sequences. A full-length copy from such an alternatively spliced PLP mRNA was identified and shown to encode a variant PLP form. All evidence suggests that this variant proteolipid is the DM-20 protein of myelin. To analyze the mechanism of alternative splicing, we determined the sequence of the corresponding part of the rat PLP gene § and found that RNA processing involves alternative 5' splice site selection in th...

The structures of two rat brain-specific 1B236 mRNAs, alternative splice products from a single gene regulated differently during postnatal brain development, were deduced from full-length cDNA clones. The 626-and 582-amino acid-long encoded proteins are indistinguishable from two forms of myelin-associated glycoprotein, a cell adhesion molecule involved in axonal-glial and glial-glial interactions in postnatal brain development, particularly in myelination. The two proteins share a single membrane-spanning domain and a glycosylated N terminus but differ in the structures of their C termini. The N terminus consists of five domains related in sequence to each other and to immunoglobulin-like molecules, especially the neural cell adhesion molecule N-CAM, suggesting a common structure for cell adhesion molecules. Rat brain protein 1B236 was originally defined by nucleotide sequence analysis of randomly selected cDNA clones of mRNAs expressed in adult rat brain but not detectable in liver or kidney (1,2). Antisera to synthetic peptides corresponding to nonoverlapping regions of the partial 1B236 sequence detected a postnatally expressed 100-kDa rat brain protein containing "30% N-linked carbohydrate and proteolytic fragments derived from its C-terminal regions (2-5). During early postnatal development, 1B236 is expressed predominantly by oligodendrocytes in myelinating fiber tracts. Subsequently, there is gradual elaboration of the adult pattern in which 1B236 mRNA is detected predominantly in subsets of neurons in grey matter regions and the 1B236 protein is restricted to specific neuronal cell bodies and fibers (refs. 2, 4, and 6; G. A. Higgins, H. Schmale, F.E.B., M. C. Wilson, R.J.M., unpublished data).We have now analyzed 1B236 expression more completely and report here the structures of two differently regulated, alternatively spliced 1B236 mRNAs that encode proteins with alternative C-terminal tails. The shared N-terminal region consists of five domains that are related in sequence to each other and to proteins of the immunoglobulin super family (8), especially the neural cell adhesion molecule N-CAM (9). We show that the 1B236 protein is indistinguishable from myelin-associated glycoprotein (MAG), a nervous system-specific glycoprotein of 100 kDa that contains -30% carbohydrate (10). MAG has been implicated in the interactions between myelinating cells and axons (10) and the formation and maintenance of the periaxonal space (11). In the peripheral nervous system, MAG appears to take over Schwann cell-axon and Schwann cell-Schwann cell interactions initiated by N-CAM and L1/neuron-glia cell adhesion molecule (12). Thus two cell adhesion molecules that act at successive stages of neural development also share significant structural properties. EXPERIMENTSPrimary Structure of the 1B236 mRNA: Alternative Splicing Produces Two Forms. We described a 1500-nucleotide (nt) partial cDNA clone of the 2500-nt 1B236 mRNA (2). Two additional clones (p1B236-18 and plB236-20) with apparently full-length inserts were...