Partial cDNA sequence to a hamster gene corrects defect in Escherichia coli pyrB mutant.
The first three enzymes of pyrimidine biosynthesis (carbamoyl-phosphate synthetase, aspartate carbamoyl-transferase, and dihydro-orotase) are carried on a multifunctional protein in mammalian cells and are on separate proteins in bacteria. A plasmid containing a cDNA sequence corresponding to 80% of a hamster mRNA for this protein was transformed into Escherichia coli mutants lacking aspartate carbamoyltransferase (pyrB) or dihydro-orotase (pyrC). Only pyrB transformants were able to grow in the absence of uracil. Plasmid recovered from primary transformants was similar in size to the original plasmid and could yield prototrophs after secondary transformation of E. coli pyrB mutants. When cell extracts were prepared from pyrB transformants, high levels of aspartate carbamoyltransferase activity were found, and the enzyme had properties identical to the mammalian enzyme, including lack of allosteric regulation, precipitation by antiserum specific to the hamster multifunctional protein, and presence of a strong aggregation center. These results demonstrate that (i) a partial hamster protein can complement E. coli defective in pyrimidine biosynthesis, (ii) the order of the enzyme domains of the multifunctional protein is likely to be NH2-dihydro-orotase-carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-COOH, and (iii) the enzyme domains appear to be self-contained at the DNA and protein levels. The protocol described here may be a general means for studying the domains of multifunctional proteins and for isolating other mammalian genes for which bacterial mutants have been prepared. It also permits study of the structure and function of the same gene in both prokaryotic and eukaryotic cells and may provide new insight into the evolution of complex genes.
D000581:Amidohydrolases / Q000235:genetics*; D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000235:genetics* / Q000302:isolation & purification; D006224:Cricetinae; D004247:DNA / Q000032:analysis*; D004080:Dihydroorotase / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D007106:Immune Sera; D008970:Molecular Weight; D009154:Mutation; D010957:Plasmids; D014169:Transformation, Bacterial
D007106:Immune Sera; D004247:DNA; D001221:Aspartate Carbamoyltransferase; D000581:Amidohydrolases; D004080:Dihydroorotase