Mapping of RNA polymerase binding sites in the H/A gene region of bacteriophage S13 by footprinting and exonuclease III analysis.

The H/A gene region of the bacteriophage S13 genome (map positions 4597 to 289) was analyzed for Escherichia coli RNA polymerase-binding sites by combined DNase I footprinting, exonuclease III analysis and DNA sequencing. Two high-affinity binding sites were identified, one corresponding to the A gene promoter at position 5384 to 56, overlapping the H/A intergenic region at position 1 to 63, the other at position 5171 to 5230, at the 3'-end of the H gene. A medium-affinity RNA polymerase binding site was mapped at position 4810 to 4850 in the middle of the H gene upstream of a low-affinity site at position 5280 to 5330. The studies complete footprinting analyses of the S13 genome from position 4597 to 2198 and in combination with previous studies on transcription provide definitive evidence on the position of the A gene promoter in S13 and the closely related phage psi X174.

Recombinant myelin-associated glycoprotein confers neural adhesion and neurite outgrowth function.

Myelin-associated glycoprotein (MAG) cDNA clones for the small (p67) and large (p72) forms were expressed in heterologous cells. Purified recombinant MAG protein was incorporated into fluorescent liposomes, and both forms were shown to bind predominantly to neurites in DRG or spinal cord cultures. This adhesion was completely blocked by Fab fragments of monoclonal anti-MAG antibody. Liposomes prepared with the control protein glycophorin or no protein failed to bind neurites. Small cerebellar neurons, which are not myelinated in vivo, failed to bind MAG liposomes. In a second test of function, p67 MAG-transfected fibroblasts were markedly enhanced in their ability to promote DRG neurite extension over a 2 day culture period compared with control fibroblasts not expressing MAG. Neurite extension was blocked by anti-MAG antibodies. These results show that both forms of MAG can facilitate the interactions between glial cells and neurites that ultimately lead to myelin formation.

Myelin-associated glycoprotein, a cell adhesion molecule of oligodendrocytes, is phosphorylated in brain.

Myelin-associated glycoprotein (MAG) has been implicated in the mediation of interactions between oligodendrocytes and neurons during the development of the myelin sheath. Here we show that MAG is phosphorylated in intact myelinating mouse brain primarily at serine residues and to a lesser extent at threonine and tyrosine residues. In vivo, only the larger of the two developmentally regulated MAG isoforms is phosphorylated. MAG can be phosphorylated at tyrosine by the v-fps and v-src protein-tyrosine kinases in vitro and by a kinase endogenous to myelin membrane preparations. MAG phosphorylated in myelin membranes in vitro also contains phosphoserine and phosphothreonine. These observations suggest that phosphorylation of MAG is physiologically significant in regulating oligodendrocyte-neuron interactions.

Mapping the B and D gene promoters of bacteriophage S13 by footprinting and exonuclease III analysis.

The region of the bacteriophage S13 genome which contains the B, K, and C genes and most of the A, D, and E genes (map positions 627 to 2198) was analyzed for Escherichia coli RNA polymerase-binding sites by combined DNase I footprinting, exonuclease III analysis, and DNA sequencing. Two high-affinity binding sites that correspond to the B and D gene promoters were mapped at positions 936 to 995 and 1779 to 1848, respectively. Positions 936 to 995 are in gene A, preceding the B gene, and positions 1779 to 1848 are in gene C, just preceding the D gene. In addition, two lower-affinity binding sites were identified at positions 1527 to 1538 and 1705 to 1756 preceding the C and D genes, respectively. The footprinting studies described here, in combination with previous studies on transcription, provide definitive evidence on the position of the B and D gene promoters in S13 and the closely related phage phi X174.

The myelin-associated glycoprotein gene: mapping to human chromosome 19 and mouse chromosome 7 and expression in quivering mice.

Myelin-associated glycoprotein (MAG), a membrane glycoprotein of 100 kDa, is thought to be involved in the process of myelination. A cDNA encoding the amino-terminal half of rat MAG has recently been isolated and sequenced. We have used this cDNA in Southern blot analysis of DNA from 32 somatic cell hybrids to assign the human locus for MAG to chromosome 19 and the mouse locus to chromosome 7. Since the region of mouse chromosome 7-known to contain several other genes that are homologous to genes on human chromosome 19-also carries the quivering (qv) locus, we considered the possibility that a mutation in the MAG gene could be responsible for this neurological disorder. While MAG-specific DNA restriction fragments, mRNA, and protein from qv/qv mice were apparently normal in size and abundance, we have not ruled out the possibility that qv could be caused by a point mutation in the MAG gene.

Molecular cloning and primary structure of myelin-associated glycoprotein.

Myelin-associated glycoprotein (MAG) may play a role in the cellular interactions leading to myelination. Using monoclonal antibodies and conventional antisera against MAG, we have isolated a cDNA clone from an expression library prepared from rat brain mRNA. The identity of the clone was confirmed by the exact match between its nucleotide sequence and two peptide sequences of 13 and 9 amino acids that we obtained by Edman degradation of two CNBr fragments of MAG. The cDNA clone hybridized to two size species of mRNA in rat approximately 3.5 kilobases in length. These mRNAs were present in brain but not liver and were expressed most abundantly at the time of active myelination (day 14). The mRNA for MAG was present at barely detectable levels in hypomyelinating jimpy mice compared to normal littermate controls. Therefore the MAG cDNA clone is both brain and myelin specific. DNA sequence analysis revealed that our MAG cDNA was derived from the same mRNA as clone p1B236, a randomly selected, brain-specific, partial cDNA isolated by Sutcliffe et al. [Sutcliffe, J. G., Milner, R. J., Shinnick, T. M. & Bloom, F. E. (1983) Cell 33, 671-682]. Analysis of the predicted protein sequence suggests that MAG has a long extracellular domain (499 amino acids), followed by a short transmembrane segment (20 amino acids) and an intracellular carboxyl-terminal domain (90 amino acids). The molecule has several glycosylation sites, three internal repeats homologous to a repeat in the neural cell adhesion molecule (N-CAM), and sites for phosphorylation near the carboxyl terminus. The primary structure reported here provides a molecular framework for further investigations into the function of the MAG molecule.