Messenger RNAs for kinesins and dynein are located in neural processes.

Abstract

The nervous systems of all jawed animals are fully myelinated. During development, the largest caliber axons are separated from smaller neighbors by oligodendrocyte (OL) processes that envelop them with compacted multilayered myelin sheaths. Usually a single OL myelinates many axon segments. Proteins used in the sheaths are synthesized at two sites, OL soma and OL processes. Myelin basic protein (MBP) and isoforms of a second small, highly basic protein, myelin-associated oligodendrocytic basic protein (MOBP) are the only proteins known to be synthesized in OL processes. The synthesis of these two proteins is ideally situated to coordinate the compaction of the cytoplasmic leaflets into the major dense line. As a prelude to their synthesis in OL processes, the mRNAs encoding MBP and MOBP must be transported to each of the sites where myelin sheaths are formed. Morphologically, these sites are thin cytoplasmic fingers, called outer tongue processes, that overlie the compacted myelin. When one homogenizes nervous tissue, these cytoplasmic fingers become entrapped in vesicles that form from compacted myelin lamellae. The resulting myelin vesicles are readily purified by subcellular fractionation. Because they trap cytoplasm derived from OL processes, they have high levels of MBP mRNA (1). In contrast, they contain relatively little of the mRNAs that originate in other neural cell compartments, including OL soma, astrocytes, neuronal soma, and their dendritic processes (2, 3). We used mRNAs purified from a low-speed supernatant (S) of homogenized rat brain and mRNAs purified from the myelin fraction (M)-material that accumulates at a 0.25 sucrose/0.85 sucrose interface-as starting materials for suppression-subtractive hybridization (4). Briefly, the S mRNA is used to make double-stranded cDNA driver, and M mRNA is used to make M cDNA tester. Both cDNAs are digested with RsaI to make small pieces. Different adaptors are ligated to each of two separate batches of digested M cDNAs. One round of hybridization is performed in which each batch of M cDNA is melted and annealed with excess S cDNA. A second hybridization is performed with the individual hybridization reactions combined. Then, the double-stranded cDNAs, which are derived from the separate testers (i.e., they have different adaptors at each end), are selectively amplified by nested PCR. All of the above protocols follow the Clontech kit manual. The PCR product, which represents mRNAs enriched in myelin, is incorporated into vector, transformed into bacteria, and colonies that represent individual cDNAs are screened by southern blot hybridization with full-