We have isolated cDNA clones encoding the four different forms of mouse myelin basic protein (MBP) and have analyzed the structure of the MBP gene. The three larger forms of MBP differ from the smallest by the inclusion of either or both of two short amino acid sequences at positions 57 and 124 of the smallest protein. The mouse genome contains a single MBP gene comprised of seven exons. The two amino acid sequences present only in the larger MBPs are encoded by separate exons. Furthermore, all exons in the coding region begin or end in complete codons so that alternative splicing does not alter the reading frame. We conclude that the four forms of this myelin protein are encoded in separate mRNAs, each derived by a simple alternative splicing of the primary MBP gene transcript. Comparison of the amino acid sequence encoded by each exon with a recent model of the secondary structure of MBP suggests that each of the seven exons encodes one or two of the predicted structural motifs of the protein.

We have isolated cDNA clones encoding three separate forms of human myelin basic protein (MBP), 21.5, 18.5, and 17.2 kDa, and have determined the nucleotide sequence of each. The three forms share a common sequence but differ by the inclusion of a 26-residue amino acid sequence near the N terminus of the 21.5-kDa protein or by the absence of an 11-residue amino acid sequence near the C terminus of the 17.2-kDa protein. The sequences either added to or deleted from the major 18.5-kDa MBP correspond exactly to exons 2 and 5 of the mouse MBP gene, suggesting that the human and mouse genes have similar exon structures. We have also identified the 21.5-kDa human MBP on immunoblots using antisera raised to a peptide encoded by the mouse exon 2 sequence. Southern blotting studies of human genomic DNA reveal a simple pattern consistent with a single human MBP gene. Thus, the three MBP mRNAs are likely to arise from alternative splicing of a primary human MBP transcript. Conservation of the 26 amino acid mouse exon 2 sequence in human MBP suggests an important role for this sequence in myelination.Myelin is a multilamellar compacted membrane structure that surrounds and electrically insulates the axon, facilitating the conduction of nerve impulses. This elaborate structure is synthesized and assembled by oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS) (1). Myelin basic protein (MBP) constitutes 30% of the total myelin protein in the CNS, but it is a lesser constituent of PNS myelin. The Unlike the case in the mouse, only a single MBP species has been identified in human myelin. The human 18.5-kDa MBP has been well characterized, and its amino acid sequence has been determined directly (6). Although minor bands, both larger (7) and smaller (8), are sometimes seen on protein gels of human MBP, these forms have not been isolated or characterized.We have examined the question of multiple forms ofhuman MBP by assembling a large collection of human MBP cDNA clones and examining their structure by restriction endonuclease mapping and nucleotide sequencing. We have identified three types of MBP cDNAs. The most common human MBP cDNA corresponds to the 18. MATERIALS AND METHODSPreparation of RNA and DNA. Total RNA was prepared from human brain by the guanidine thiocyanate/cesium chloride method (9). Fractions enriched in mRNA were prepared from total RNA by oligo(dT) selection (10). RNA was fractionated for blot hybridization in agarose/formaldehyde gels (10). High molecular weight DNA was prepared from human placenta.

Shiverer (shi) is an autosomal recessive mutation in the mouse characterized by an almost total lack of central nervous system myelin. While small amounts of other myelin components are present in the brain of the shi mouse, the four forms of myelin basic protein (MBP) are not detectable. Previous investigations by us and others indicate that the MBP gene has undergone a major rearrangement in the shi mutant. Herein, we report in detail the nature and extent of the rearrangement: a 20-kilobase region within the MBP gene is missing in the mutant. We map the 5' breakpoint ofthe deletion to the second intron and the 3' breakpoint to a site 2 kilobases beyond the last MBP exon. The junction of the upstream and downstream portions of the gene contains only one nucleotide not accounted for by the wild-type MBP gene sequence. The 3' side of the deletion occurs in the 3rd of 11 tandem repeats of a 31-base-pair sequence. This region is rich in alternating purine and pyrimidine stretches, sequences that have been associated with both Z-DNA structures and gene rearrangements. Mammalian oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS) produce a highly specialized multilamellar myelin membrane that surrounds the neuronal axons and greatly facilitates conduction of electrical impulses (reviewed in ref. 1). The myelin of the CNS has a relatively simple composition in which a family of closely related myelin basic proteins (MBPs) accounts for 30-40% of the total protein in the sheath (reviewed in ref. 2). Peripheral nervous system myelin contains a smaller and dispensable amount of the same MBPs (3, 4). Recent work by us (5) and others (6) has shown that a single gene encodes the family of MBPs and that the various forms of the MBPs are generated by a mechanism of alternative splicing of the mRNA.A number of mutations have been identified in mice that affect oligodendrocyte function and/or Schwann cell function and, consequently, lead to dysmyelination in the CNS and/or the PNS (reviewed in ref. 7). The shiverer (shi) mutation is autosomal recessive and is characterized by the onset of tremors at about the 12th day of life, seizures at later times, and a progressive deterioration ending in an early death (8, 9). The mutation principally affects oligodendrocyte functions and the CNS of the shi mice is almost entirely devoid of myelin membrane (10). The normal amount of myelin is present in the PNS of these animals, but differences in its structure have been noted (4,11,12). The primary defect in shi mice appears to be the absence of MBP (13). By radioimmune assay, MBP levels in shi mice are <0.1% that of wild type (14).The shi allele has been mapped to chromosome 18 by Sidman et al. (15) using classic genetic techniques. The gene encoding the MBP family has also been mapped to chromosome 18 by in situ hybridization and Southern blot analysis of DNA isolated from mouse-hamster somatic cell hybrids (16). Recent work by Roach et al. (17,18) and by us (5) demonst...

A mixed culture dechlorinating 1,2-dichloroethane (1,2-DCA) to ethene was enriched from groundwater that had been subjected to long-term contamination. In the metagenome of the enrichment, a 7-kb reductive dehalogenase (RD) gene cluster sequence was detected by inverse and direct PCR. The RD gene cluster had four open reading frames (ORF) showing 99% nucleotide identity with pceB, pceC, pceT, and orf1 of Dehalobacter restrictus strain DSMZ 9455 T , a bacterium able to dechlorinate chlorinated ethenes. However, dcaA, the ORF encoding the catalytic subunit, showed only 94% nucleotide and 90% amino acid identity with pceA of strain DSMZ 9455 T . Fifty-three percent of the amino acid differences were localized in two defined regions of the predicted protein. Exposure of the culture to 1,2-DCA and lactate increased the dcaA gene copy number by 2 log units, and under these conditions the dcaA and dcaB genes were actively transcribed. A very similar RD gene cluster with 98% identity in the dcaA gene sequence was identified in Desulfitobacterium dichloroeliminans strain DCA1, the only known isolate that selectively dechlorinates 1,2-DCA but not chlorinated ethenes. The dcaA gene of strain DCA1 possesses the same amino acid motifs as the new dcaA gene. Southern hybridization using total genomic DNA of strain DCA1 with dcaA gene-specific and dcaB-and pceB-targeting probes indicated the presence of two identical or highly similar dehalogenase gene clusters. In conclusion, these data suggest that the newly described RDs are specifically adapted to 1,2-DCA dechlorination.Chlorinated alkanes are prevailing groundwater contaminants in many industrialized countries (www.epa.gov/enviro /html/tris/ez.html), and they cause serious environmental problems (14). Among these, 1,2-dichloroethane (1,2-DCA) represents one of the world's most important toxic C 2 chlorinated aquifer pollutants. From 1987 to 1993, over 209 tons of 1,2-DCA were released into groundwater (www.epa.gov/enviro /html/tris/ez.html). Enhancement of natural attenuation processes can play a major role in achieving remediation, where applicable (17, 30), and often bioremediation alone or in combination with physical treatment of the most contaminated areas could represent the most convenient solution. The main catalyzers of the bioremediation processes are microorganisms that can dehalogenate and/or mineralize the toxic molecules with their enzymatic systems (12,40).A metabolic process of particular interest is based on dehalorespiration, a reaction that couples reductive dehalogenation with energy conservation (11,35,40). Dehalorespiration is one of the key processes for the remediation of polluted groundwaters (20,24,25). The key catalysts in dehalorespiration are reductive dehalogenases (RDs), membrane-associated enzymes with low levels of nucleotide identity but with some common traits, such as two iron-sulfur clusters as prosthetic groups, a twin-arginine translocation signal peptide (TAT system), and corrinoid cofactors (22). Recently, it has been reported that ...

No abstract