Isolation by genetic complementation of two differentially expressed genes for beta-isopropylmalate dehydrogenase from Aspergillus niger.

We have constructed an Aspergillus niger cDNA library with a yeast expression vector. The library DNA complemented a leucine auxotroph of Saccharomyces cerevisiae (strain BWG1-7a) at a frequency of 4x10(-4). Plasmids rescued from the yeast prototrophs also complemented Escherichia coli (strain MC1066) deficient in leucine biosynthesis. Sequence determination of the rescued plasmids revealed two genes for beta-isopropylmalate dehydrogenase, which we called leu2A and leu2B. Genomic-blot analysis suggested that both leu2A and leu2B were derived from single-copy genes. Northern-blot hybridization showed that in nutrient-rich medium a leu2A transcript accumulated during germination and log-phase growth while the leu2B transcript appeared late in the growth phase. In minimal medium, only leu2A expression was greatly stimulated. We examined the codon preference of these two genes. Whereas leu2A shows a bias in codon usage typical of A. niger genes, leu2B does not. These results indicate the presence in A. niger of two highly divergent, differentially regulated, isozymes for beta-isopropylmalate dehydrogenase.

Heterogeneity in cblG: differential retention of cobalamin on methionine synthase.

Cultured fibroblasts from patients with functional methionine synthase deficiency have been shown to belong to two complementation classes, cblE and cblG. Both are associated with decreased intracellular levels of methylcobalamin (MeCbl) and decreased incorporation of label from 5-methyltetrahydrofolate into macromolecules. Methionine synthase specific activity is normal or near normal in cell extracts from cblE patients under standard reducing conditions, whereas specific activity is low in cblG extracts. Seven of 10 cblG cell lines accumulated [57Co]CN-Cbl equivalent to control cells and showed similar proportions of label associated with the two intracellular cobalamin binders, methionine synthase and methylmalonyl-CoA mutase. The remaining three cblG lines showed reduced accumulation of labeled Cbl and virtually none associated with methionine synthase. The specific activity of methionine synthase was decreased in cell extracts from both cblG subgroups, being almost undetectable in extracts from the latter three lines. Incorporation of label from [14C]MeTHF into either macromolecules or into methionine was decreased in both cblG groups, but was paradoxically higher in the three lines with very low in vitro methionine synthase activity. These results demonstrate further heterogeneity within cblG and suggest that the defect in the three variant lines affects the ability of methionine synthase to retain Cbl.

Changes in cobalamin metabolism are associated with the altered methionine auxotrophy of highly growth autonomous human melanoma cells.

Our aim was to identify the biochemical defect responsible for the inability of highly growth autonomous human tumor cells to proliferate in culture medium devoid of methionine, but containing homocysteine and 5-methyletrahydrofolic acid. We have adopted the terms "homocysteine-responsive" and "homocysteine-nonresponsive" to describe cells which can or cannot proliferate in methionine-free homocysteine-supplemented medium. Using a panel of genetically related homocysteine-responsive and -nonresponsive human melanoma cell lines, the results from a number of experiments indicate that acquisition of the "homocysteine-nonresponsive phenotype" is associated with the reduced intracellular accumulation of methyl-cobalamin, a critical cofactor of the methionine synthase enzyme. When in vitro methionine synthase assays were performed in the presence of exogenously added methyl-cobalamin, specific methionine synthase activity in extracts obtained from homocysteine-responsive cells was only twofold greater than that observed with extracts prepared from homocysteine-nonresponsive cells. However, when exogenous methyl-cobalamin was omitted from the enzyme assays, methionine synthase activity in extracts derived from homocysteine-nonresponsive cells was dramatically reduced, compared with the small decrease observed with homocysteine-responsive cell extracts. Compared with their homocysteine-responsive counterparts, homocysteine-nonresponsive cells exhibited increased levels of cobalamin efflux and decreased intracellular accumulation of methyl-cobalamin. There was a clear relationship between the abilities of these related melanoma cell lines to proliferate in methionine-free homocysteine-supplemented medium, and the extent of cobalamin loss and capacity of exogenously added methyl-cobalamin to stimulate in vitro methionine synthase activity. These results indicate a link between alterations in the intracellular trafficking and/or metabolism of cobalamin and the increased growth autonomy of human melanoma cells.