Partial cDNA sequence to a hamster gene corrects defect in Escherichia coli pyrB mutant.
- 1 November 1983
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 80 (22), 6897-6901
- https://doi.org/10.1073/pnas.80.22.6897
Abstract
The first 3 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 c[complementary]DNA sequence corresponding to 80% of a hamster mRNA for this protein was transformed into E. 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. A partial hamster protein can complement E. coli defective in pyrimidine biosynthesis. The order of the enzyme domains of the multifunctional protein is likely to be NH2-dihydro-orotase-carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-COOH. 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 were prepared. The structure and function of the same gene can be studied in both prokaryotic and eukaryotic cells and may provide new insight into the evolution of complex genes.Keywords
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