Genetic control of cobalamin binding in normal and mutant cells: Assignment of the gene for 5-methyltetrahydrofolate:L-homocysteine S-methyltransferase to human chromosome 1 |
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| Authors: | Ira S. Mellman Pin-Fang Lin Frank H. Ruddle Leon E. Rosenberg |
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| Affiliation: | Department of Human Genetics, Yale University, 333 Cedar Street, New Haven, Connecticut 06510;Department of Biology, Yale University, 333 Cedar Street, New Haven, Connecticut 06510 |
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| Abstract: | When extracts prepared from cultured human or rodent fibroblasts grown in medium containing [(57)Co]cobalamin were analyzed by polyacrylamide gel electrophoresis, most of the intracellular radioactivity migrated with the activity of the cobalamin-dependent enzyme 5-methyltetrahydrofolate:L-homocysteine S-methyltransferase (EC 2.1.1.13). Because the rodent and human forms of this enzyme are electrophoretically different, we used the binding of [(57)Co]cobalamin to detect the presence of the human methyltransferase isozyme in rodent-human somatic cell hybrids. As expected, binding and methyltransferase activities were found to cosegregate, thus confirming genetically their electrophoretic identity. Accordingly, we examined the [(57)Co]cobalamin-binding patterns and human chromosome contents of a panel of 12 rodent-human hybrid clones, and concluded that the gene for the methyltransferase (designated Mtr) is located on human chromosome 1. Using this information, we probed the nature of the molecular defect exhibited by fibroblasts cultured from patients expressing the cbl C mutation. Although these cells are unable to associate newly taken up [(57)Co]cobalamin with the methyltransferase, hybrids of mouse L-cells and cbl C cells containing chromosome 1 show a "reappearance" of the human [(57)Co]cobalamin-methyltransferase. These results indicate that the cbl C mutation does not affect the methyltransferase apoprotein, but rather some metabolic step that must convert cobalamin to a chemical form capable of attaching to the enzyme. |
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