ABSTRACT
We have characterized 202 lacI(-) mutations, and 158 dominant lacI(-d) mutations following treatment of Escherichia coli strains NR6112 and EE125 with 1-nitroso-6-nitropyrene (1,6-NONP), an activated metabolite of the carcinogen 1,6-dinitropyrene. In all, 91% of the induced point mutations occurred at G:C residues. The -(G:C) frameshifts were the dominant mutational class in the lacI(-) collections of both NR6112 and EE125, and in the lacI(-d) collection of NR6112. Frameshift mutations occurred preferentially in runs of guanine residues, and their frequency increased with the length of the reiterated sequence. In strain EE125, which contained the plasmid pKM101, there was a marked stimulation in the frequency of base substitution mutations that was particularly apparent in the lacI(-d) collection. This study completes a comprehensive analysis of 1194 lacI(-) and 348 lacI(-d) mutations induced by either 1,6-NONP or its positional isomer 1-nitroso-8-nitropyrene (1,8-NONP) in strains of E. coli that differ with regard to their ability to carry out nucleotide excision repair and/or their ability to express the translesion synthesis DNA polymerase RI (MucAB) encoded by plasmid pKM101. Among the mutations are 763 frameshift mutations, 367 base substitutions and 47 deletions; these mutations have been characterized at more than 300 distinct sites in the lacI gene. Our studies provide detailed insight into the DNA sequence alterations and mutational mechanisms associated with dinitropyrene mutagenesis. We review the mutational spectra, and discuss cellular lesion repair or tolerance mechanisms that modulate the observed mutational specificity.
Subject(s)
Bacterial Proteins/genetics , Escherichia coli Proteins , Escherichia coli/drug effects , Escherichia coli/genetics , Genes, Bacterial , Mutagens/toxicity , Mutation , Nitroso Compounds/toxicity , Pyrenes/toxicity , Repressor Proteins/genetics , Amino Acid Substitution , Bacterial Proteins/metabolism , Base Sequence , Carcinogens/metabolism , Carcinogens/toxicity , DNA Repair , Escherichia coli/metabolism , Frameshift Mutation , Lac Repressors , Plasmids/genetics , Point Mutation , Pyrenes/metabolismABSTRACT
To establish structure-function relationships for human (h) LH, 14 murine monoclonal antibodies (MABs) to hLH were characterized in terms of their affinity of binding (Ka), their specificity for intact glycoproteins and their subunits, their paratopic relationships, and their ability to interfere with the biological activity of hLH. The Ka values obtained ranged between 2.4 x 10(6) and 1.0 x 10(10) for intact hLH and between 2.3 x 10(6) and 7.5 x 10(8) liters/M for the free alpha- and beta-subunits, indicating that, in general, the antibodies showed higher avidity for the intact hormone. Six MAB recognized both the intact and free alpha-subunit of hLH, cross-reacted with intact hCG, hFSH, and hTSH, and thus appeared to be alpha-directed. Four MAB were beta-directed, recognizing both intact hLH and its free beta-subunit. One of these beta-directed MABs also cross-reacted with intact hCG, hFSH, and hTSH, while two others recognized both intact and free beta-subunits of hLH and hCG. The fourth beta-directed MAB was quite specific for intact hLH and its beta-subunit. The remaining four MABs recognized epitopes only on the intact hormone; three recognized intact hLH and hCG, and the fourth was specific for intact hLH. Their paratopic relationships tested in competitive binding studies resulted in either mutual competition or complementarity, sometimes with cooperativity. Biointerference, defined as the ability to inhibit hLH-induced testosterone biosynthesis in dispersed rat Leydig cells, indicated that three of the alpha- and one of the beta-directed antibodies neutralized the biological response of hLH in this bioassay in a dose-responsive manner. Their ability to inhibit hLH bioactivity largely paralleled their affinity constants. Our data have allowed us to establish a tentative topographic relationship of epitopes to the biological region of the molecule of hLH, foreshadowing (in additive binding studies) some of the possible combinations of antibodies that might allow us to design two- or multiple-site immunometric assays in which measurement of immunoactive LH reflects biological activity. In addition, these studies suggest that both the alpha- and beta-subunits participate in LH receptor binding and/or biological activity.
