Guanine auxotrophs of Escherichia coli were isolated following mutagenesis by N-methyl-N′-nitro-N-nitrosoguanidine or ethyl methanesulphonate. The mutants were classified according to growth properties and absence of IMP dehydrogenase or GMP synthetase activity. Mutations in guaB (IMP dehydrogenase-less) were analysed by reversion and suppression tests; all were of the base substitution missense type except for one possible frameshift and one polar nonsense mutation. GuaB mutants were examined for protein (CRM) that cross-reacts with monospecific antibodies to IMP dehydrogenase; approximately half were CRM+. Enzyme complementation in vitro was detected in mixed denatured and renatured cell-free extracts of any CRM+guaB mutant and PL1138 (guaB105, CRM+); CRM− mutants did not complement. GuaB105 maps distal to all other guaB mutations except guaB86 (CRM−). Two hybrid enzymes produced by complementation were less stable to heat than native IMP dehydrogenase, although kinetic constants were similar. These observations indicate interallelic complementation between guaB mutants and are consistent with the demonstration of identical subunits for IMP dehydrogenase (Gilbert et al., 1979). Only the subunits supplied by PL1138 are catalytically active in the hybrid enzymes suggesting that this mutant may produce a repairable polypeptide whereas the enzymes of complementing mutants may be defective at the active site.
BrettM.,
ChambersG. K.,
HolderA. A.,
FinchamJ. R. S.1976; Mutational amino acid replacements in Neurospora crassa NADP-specific glutamate dehydrogenase. Journal of Molecular Biology 106:1–22
GilbertH. J.,
DrabbleW. T.1978; Active-site alkylation of IMP dehydrogenase from wild-type Escherichia coli K12 and some guaB mutants. Proceedings of the Society for General Microbiology 5:49–50
GilbertH. J.,
LoweC. R.,
DrabbleW. T.1978; The subunit structure of IMP dehydrogenase of Escherichia coli K12. Proceedings of the Society for General Microbiology 5:23
KidaS.,
CrawfordI. P.1974; Complementation in vitro between mutationally altered β2 subunits of Escherichia coli tryptophan synthetase. Journal of Bacteriology 118:551–559
KrishnaiahK. V.1975; Inosinic acid 5′-monophosphate dehydrogenase from Escherichia coli: purification by affinity chromatography and some properties. Archives of Biochemistry and Biophysics 170:567–575
LambdenP. R.,
DrabbleW. T.1973; The guaoperon of Escherichia coli K-12: evidence for polarity from guaB to gua A
. Journal of Bacteriology 115:992–1002
MacpheeD. G.,
StockerB. A. D.1969; Suppression of amber and ochre mutants in Salmonella typhimurium by a mutant F′-l-gal factor carrying an ochre suppressor gene. Journal of Bacteriology 100:240–246
MagerJ.,
MagasanikB.1960; Guanosine 5′-phosphate reductase and its role in the interconversion of purine nucleotides. Journal of Biological Chemistry 235:1474–1478
NijkampH. J. J.,
DehaanP. G.1967; Genetic and biochemical studies of the guanosine 5′-monophosphate pathway in Escherichia coli
. Biochimica et biophysica acta 145:31–40
SchaferM. P.,
HannonW. H.,
LevinA. P.1974; In vivo and in vitro complementation between guaB and in vivo complementation between gua Aauxotrophs of Salmonella typhimurium. Journal of Bacteriology 117:1270–1279
SchlesingerM. J.1974; Variants of Escherichia coli alkaline phosphatase: an example of the role of genetics in a study of protein structure and function. Biochemical Society Transactions 2:827–831
UllmanA.,
JacobF.,
MonodJ.1967; Characterisation by in vitro complementation of a peptide corresponding to an operator-proximal segment of the β-galactosidase structural gene of Escherichia coli. Journal of Molecular Biology 24:339–343
VogelH. J.,
BonnerD. M.1956; Acetylornithinase of Escherichia coli: partial purification and some properties. Journal of Biological Chemistry 218:97–106