Xgt11 cDNA libraries derived from human brain were screened with oligonucleotide probes for recombinants that code for a subunits of G signal transduction proteins. Eleven a, clones were detected with both probes and characterized. Four types of a, cDNA were cloned that differ in nucleotide sequence in the region that corresponds to amino acid residues 71-88. The clones differ in the codon for as amino acid residue 71 (glutamic acid or aspartic acid), the presence or absence of codons for the next 15 amino acid residues, and the presence or absence of an adjacent serine residue. S1 nuclease protection experiments revealed at least two forms of a8 mRNA. A mechanism for generating four species of a8 mRNA by alternative splicing of precursor RNA is proposed.Membrane-associated guanine nucleotide binding proteins (G proteins) act as signal transducers, coupling receptors for light, hormones, or neurotransmitters to effectors such as adenylate cyclase or cGMP phosphodiesterase, and possibly ion channels (1, 2). Known G proteins include Gs and G1 required for receptor-mediated activation or inhibition, respectively, of adenylate cyclase, two species of transducin (TD)-one in rod photoreceptor outer segments (3), the other in cones (4, 5)-and G0, a G protein of unknown function, abundant in brain (1). There is immunochemical (6) and functional (7) evidence suggesting the existence of additional G proteins. Known G proteins are composed of three protein subunits, a, /3, and y; the a subunits bind guanine nucleotides, catalyze GTP hydrolysis, and couple, directly or indirectly, receptors with effector molecules (1). Comparison of the amino acid sequences of different species of G a-subunits shows that sequences are highly conserved in some, but not all, regions of the protein (8) and that a-subunits are related to the ras family of proteins (8) AAGATGATGGCGGTCACGTCCTCGAAGCCGT-GGATCCACTTCTT 3'). Each probe (='1.5 x 106 cpm/ml, 175 fmol/ml) was added to a set of replicate filters and incubated for 16 hr at 420C. Each filter was washed three times in a solution containing 60 mM NaCl/6 mM sodium citrate and 0.1% NaDodSO4 at 230C for 20 min per wash, washed once at 420C in 60 mM NaCl/6 mM sodium citrate and 0.1% NaDodSO4 for 3 min, and then subjected to autoradiography. Insert DNA was excised with EcoRI endonuclease and subcloned into M13mpl8. Additional DNA fragments were obtained by incubating insert DNA with BamHI or Hae III endonuclease and subcloning into M13mpl8 or M13mpl9.Nucleotide sequences were obtained by the dideoxynucleotide chain-termination method (16). Maxam-Gilbert sequencing (17) was used to clarify ambiguous sequences.Total RNA for transfer blots was prepared (18) (G,) or inhibit (Gi) adenylate cyclase; aTD, a-subunit of transducin, a G protein of rod photoreceptor cells that activates cGMP phosphodiesterase; a0, a-subunit of G., a G protein of unknown function; TD, transducin; bp, base pair(s). 8893The publication costs of this article were defrayed in part by page charge payment. This article...

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.

ABSTRACTcDNA clones encoding proteolipid protein (PLP) were isolated from a mouse brain library and sequenced. We describe two transcripts arising from the PLP locus by alternative splicing: the major one encodes the 277-amino acid PLP protein and the minor one corresponds to the DM-20 protein, a PLP-like protein of 20,000 Mr that shares both amino and carboxyl regions with PLP. These two transcripts lack -70 bases in PLP mRNA from the dysmyelinating jimpy mutant. The deletion spans amino acids 208-232; however, this region is present in thejimpy PLP-encoding gene. We propose that the jimpy mutant suffers a point mutation or the deletion of a few bases in the PLP gene that alters the normal splicing pattern and generates partially deleted PLP transcripts. RESULTS AND DISCUSSIONConservation of PLP Sequence. PLP is remarkably well conserved among mouse (this report), rat (1, 16), cow (2), and human (C.P., L.D.H., and R.A.L., unpublished work) at the amino acid and nucleotide level. The protein encoded by the mouse PLP cDNA (Fig. 1) is identical to the rat (16) and human (C.P., L.D.H., and R.A.L., unpublished work) PLP and displays only two conservative amino acid differences from the bovine sequence (2). The striking degree of conserAbbreviations: PLP, proteolipid protein; MBP, myelin basic protein.

Myelin is a highly specialized membrane unique to the nervous system that ensheaths axons to permit the rapid saltatory conduction of impulses. The elaboration of a compact myelin sheath is disrupted in a diverse spectrum of human disorders, many of which are of unknown etiology. The

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.