I developed a radioimmunoassay for P0, the major peripheral myelin protein, and adapted an existing radioimmunoassay for myelin basic protein. Results from these assays showed that Schwann cells do not make either protein before myelination begins, and accumulate P0 and myelin basic protein with the same time course during development. Schwann cells cultured in the absence of neurons do not express detectable levels of either protein, but they do continue to synthesize sulfatide, a myelin sulfolipid, apparently indefinitely, as shown by biosynthetic labeling.
The trembler (Tr/+) mouse mutant has a pronounced reduction in peripheral myelin, while central myelin and peripheral unmyelinated nerves appear normal. This myelin deficiency is caused by an autonomous Schwann cell defect. Since the Trembler phenotype is expressed in heterozygotes, most previous studies were confined to Tr/+ mice. Using two alleles, I show here that the six possible genotypes can be ordered as follows: +/+ > Trj/+ > Tr/+ > Trj/Trj > Trj/Tr > Tr/Tr, based on myelin basic protein radioimmunoassays of sciatic nerve extracts. The amount of compact myelin in each genotype, as shown by electron microscopy, is in good agreement with the radioimmunoassay results, with two important provisos. First, Tr/Tr mice have 1% of wild-type myelin basic protein levels but essentially no compact myelin. Secondly, the first steps in the above genetic series reduce both the average myelin sheath area and the number of myelin sheaths by similar amounts, while the last steps primarily reduce the number of myelin sheaths. These results suggest that, if the activity of the Tr+ gene product is reduced below a certain point, myelin protein synthesis can be induced without forming a myelin sheath. Visualization of P0 by indirect immunofluorescence shows that Schwann cells without compact myelin express much lower levels of P0 than adjacent Schwann cells, associated with the same axon, that do form compact myelin. Finally, although Trembler Schwann cells proliferate excessively in vivo, their proliferation is not affected by Tr gene dose. In vitro Trj/Trj Schwann cell proliferation is apparently normal. Thus, it is likely that the function of the Tr+ gene product is not directly concerned with Schwann cell proliferation.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Revel, Jean-Paul}, } @phdthesis{10.7907/bhjv-8053, author = {Nicholson, Bruce John}, title = {Biochemistry and Diversity of the Gap Junction Protein: A Study of Liver, Heart and Lens}, school = {California Institute of Technology}, year = {1983}, doi = {10.7907/bhjv-8053}, url = {https://resolver.caltech.edu/CaltechTHESIS:10222019-124810283}, abstract = {Fractions highly enriched for gap junctions by morphological criteria have been isolated from rat liver, heart and eye lens, although some question exists as to the nature of the structures from lens. The junctions from each tissue are comprised of a single major protein of Mr 28,000 in the liver, Mr 30,000 in the heart, and Mr 26,000 (MIP 26) in the lens. The polypeptide profile of the liver fraction is complicated by endogenous proteolysis and aggregation in SDS of the gap junction protein and the presence of about 20% non-junctional material. Heart and lens junction proteins are also found to aggregate in SDS, while endogenous proteolysis typically reduces the cardiac gap junction protein to Mr 28,000 during isolation.
Comparisons of two-dimensional peptide maps of the junctional proteins from these tissues, and the use, where necessary, of a third dimension of resolution (HPLC), demonstrates the three proteins to be very different in terms of their primary structures. The protein of each tissue, however, seems well conserved between mammalian species. For liver and lens, this finding has been confirmed in amino acid analyses and partial NH2-terminal sequences (to 58 and 33 residues, respectively). Cleavage products of these two proteins have also been produced to allow further sequence analysis in the future. In spite of the differences in primary structure, some conservation of the tertiary structures of these proteins is suggested by proteolysis of intact junctions (likely restricted to the cytoplasmic surfaces). Liver and heart gap junction proteins are reduced by trypsin to two fragments of Mr 10,000, while a single Mr 21,000 fragment is produced from lens MIP 26. Sequence analysis (liver and lens only) indicates that most of the protein removed by tryptic hydrolysis is from the carboxy-terminus, although an additional loop of 4,000 daltons is excised from the center of the liver polypeptide and five residues are lost from the NH2-terminus of the lens protein.
The extent and possible significance of this surprising tissue specificity of the gap junction protein are discussed in the light of these findings.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Revel, Jean-Paul}, }