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N. Y. S. Institute for Basic Research in Developmental Disabilities, Staten Island, NY
Oligodendrocytes, the myelin-forming cells in the CNS, synthesize proteins in two distinct locations: the cell body, and in each process where myelin sheaths form. Morphologically these "outer tongue" processes are cytoplasmic channels that run along the outer surface of each myelin sheath. In mammals, myelin basic protein (MBP), a major constituent of compact myelin, is synthesized in these processes and moves rapidly (within minutes) into compact myelin. Proteins synthesized in the cell body take roughly 30 minutes to incorporate into compact myelin.
To place MBP in each sheath, oligodendrocytes synthesize protein in many (up to 40, [1]) cytoplasmic processes. We recently identified many other proteins (nearly 100, based on cDNA sequences representing mRNAs enriched in myelin) that are synthesized in rat oligodendrocyte processes using a combination of subcellular fractionation and suppression subtractive hybridization (2,3). To broaden our understanding of the role that local protein synthesis plays in myelination, we applied the same approach to identify proteins synthesized in oligodendrocyte processes of an elasmobranch, the spiny dogfish (Squalus acanthias). We already found (Gould, unpubl.) that MBP was not synthesized in dogfish oligodendrocyte processes by in situ hybridization, since the mRNA is retained in the oligodendrocyte soma and not transported to the cells’ processes.
We prepared "driver" and "tester" cDNAs from total homogenate and myelin for the subtractive hybridization experiment. Briefly, three female spiny dogfish were killed with an overdose of anesthetic. Their brains were removed and homogenized in a buffered hyperosmotic sucrose (1.2 M) solution; previously we had found that mRNAs located in oligodendrocyte processes are trapped more effectively in myelin vesicles homogenized with hyperosmotic homogenization solution (3). A portion of the homogenate was extracted for RNA (represents the entire population of RNAs in the dogfish brain and is the source of "driver") with TRI reagent (Molecular Research Center). Buffer was added to the remaining homogenate to reduce the osmolarity to 0.85 M sucrose, the sample was placed in an ultracentrifuge tube, overlaid with 0.25 M sucrose, and centrifuged (100,000 x g for 3.5 h). Myelin vesicles floating on the 0.85 M sucrose were collected, and myelin fraction RNA was prepared with TRI reagent (Molecular Research Center) and used to prepare "tester." Messenger RNA was prepared from both homogenate and myelin fraction RNAs (MicroPoly(A) PuristTM mRNA purification kit, Ambion). Homogenate and myelin mRNAs were then converted to "driver" and "tester" cDNA, and a subtraction product (enriched in cDNAs that represent mRNAs enriched in myelin) was prepared with PCR-SelectTM cDNA Subtraction Kit (CLONTECH) according to the manufacturer’s protocol. Several products were amplified by PCR, subcloned into pGEM T Easy vector (Promega), and clones were taken to prepare plasmids (minipreps). The cDNAs were sequenced in the Josephine Bay Paul Center for Comparative Molecular Biology and Evolution at the Marine Biological Laboratory in Woods Hole, Massachusetts.
In all, 74 sequences were analyzed (BLAST (N) search of the GenBank non-redundant database) (Table 1). Unlike rat cDNAs (prepared in the same fashion), which mainly represented MBP and myelin-associated oligodendrocytic basic protein (MOBP) mRNAs (1), none of the dogfish cDNA represented MBP or MOBP homologs. As with rat, about half were unrelated to mRNAs in the current GenBank database, and high portions were derived from mitochondrial DNA. Only four of the known sequences, ß-catenin, proopiomelanocortin (POMC), heat shock protein A5 (HspA5), and dihydropyrimidinase-like protein, matched sequences in the GenBank database throughout. The portions of HspA5 that matched the human sequence were 3'-coding. The non-coding portion was less conserved. Small portions of five other cDNAs—SINE, ß-spectrin, ribosomal protein L1, evx2, and Ig heavy chain—matched known sequence in the GenBank database.
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This study was supported by grants from the National Multiple Sclerosis Society (RMG) and the National Science Foundation (RS and RMG).
Footnotes
1 Marine Models in Biological Research Program, Woods Hole, MA. 
2 N. Y. S. Institute for Basic Research in Developmental Disabilities, Staten Island, NY.
3 Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, MA.
Literature Cited
This article has been cited by other articles:
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  H. B. Werner, K. Kuhlmann, S. Shen, M. Uecker, A. Schardt, K. Dimova, F. Orfaniotou, A. Dhaunchak, B. G. Brinkmann, W. Mobius, et al. Proteolipid Protein Is Required for Transport of Sirtuin 2 into CNS Myelin J. Neurosci., July 18, 2007; 27(29): 7717 - 7730. [Abstract] [Full Text] [PDF]
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 N. Pahl, S. Homsi, H. G. Morrison, and R. M. Gould mRNAs Located in Squalus acanthias (Spiny Dogfish) Oligodendrocyte Processes Biol. Bull., October 1, 2002; 203(2): 217 - 218. [Full Text] [PDF]
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