Monoclonal antibodies specific for sickle cell hemoglobin.

Two mouse hybridoma cell lines were isolated which produce monoclonal antibodies that bind hemoglobin S. The mice were immunized with peptide-protein conjugates to stimulate a response to the amino terminal peptide of the beta chain of hemoglobin S, where the single amino acid difference between A and S occurs. Immunocharacterization of the antibodies shows that they bind specifically to the immunogen peptide and to hemoglobin S. The specificity for S is high enough that one AS cell in a mixture with a million AA cells is labeled by antibody, and such cells can be analyzed by flow cytometry. Immunoblotting of electrophoretic gels allows definitive identification of hemoglobin S as compared with other hemoglobins with similar electrophoretic mobility.

On the enzymatic basis for mutagenesis by manganese.

The effects of manganese on DNA synthesis fidelity are measured using T4 DNA polymerase. When the nucleotide analogue 2-aminopurine deoxyribonucleoside triphosphate competes against dATP at thymine sites on template DNA, the aminopurine misincorporation frequency increases from 6.3% in the presence of Mg2+ to 29.2% in the presence of Mn2+. The major cause of the increased error rate is an approximate 4-fold increase in the frequency of aminopurine misinsertions. Exonucleolytic proofreading of aminopurine is similar in the presence of Mn2+ and Mg2+. However, the excision frequency of the correct nucleotide, dAMP, is increased 2-fold with Mn2+. In experiments in which insertion and incorporation velocities of aminopurine and adenine are measured independently of each other, a 5- to 10-fold decrease in the Michaelis constant for aminopurine is observed in the presence of Mn2+ compared to a 2-fold decrease in the Km for adenine. In contrast to the marked differential reduction in the ratio of aminopurine to adenine Km values, the maximum insertion velocities of both nucleotides are reduced by similar amounts (40-fold). We suggest that the mutagenic action of Mn2+ can be attributed primarily to a significant differential increase in binding of mispaired relative to correctly paired nucleotides to the polymerase-template complex. The resulting increase in the ratio of residence times for mispaired compared with correctly paired nucleotides on the complex results in their increased frequency of misinsertion. A smaller contributing factor to Mn2+-induced mutagenesis is a loss of proofreading specificity. We propose that the losses in both the specificities of nucleotide insertion and excision (proofreading) share a common molecular origin in which nucleotides are bound in the presence of Mn2+ in distorted configurations at the polymerase insertion and excision active sites resulting in increased nonspecific enzyme-substrate binding forces at the expense of template-substrate base pair specific hydrogen bonds.

Passive polymerase control of DNA replication fidelity: evidence against unfavored tautomer involvement in 2-aminopurine-induced base-transition mutations.

We consider the role of unfavored tautomers in causing base-substitution transition mutations. Data obtained with the base analogue 2-aminopurine (AP) for the frequency of forming AP.T and AP.C base mispairs can be shown to be in probable conflict with tautomer model predictions. An alternative model, in which individual hydrogen bonds exhibit different bond strengths depending upon their ring position, is proposed to account for the frequencies of forming correct and incorrect base pairs. In this "differential H-bonding" model, disfavored tautomers of AP and those of common nucleotides play a generally insignificant role. A hydrogen-bonding free energy scale is derived in which free energy differences are obtained for all possible matched and mismatched base pairs. We also show that recent in vitro data for the formation of AP.C base pairs are consistent with a "passive polymerase" theoretical model in which base selection is governed not by the enzyme but by differences in base-pairing free energies.

Cell sorter immunofluorescence detection of human erythrocytes labelled in suspension with antibodies specific for hemoglobin S and C.

We have developed an immunochemical method for labeling human red blood cells in suspension with hemoglobin-specific antibodies. A membrane permeable cross-linking reagent, dimethyl suberimidate, is used to covalently bind, in situ, a fraction of the intracellular hemoglobin to integral membrane proteins. Hypotonic lysis and washing of the cells removes the unbound hemoglobin resulting in red blood cell ghosts which are permeable to macromolecules. Fluorescein-labeled antibodies for the hemoglobin variants S and C bind specifically to hemoglobin AS and AC ghosts, respectively, and not to normal hemoglobin AA ghosts. This technique can be used to prepare ghost suspensions for cell sorter analysis in which large numbers (10(9)--10(10)) of normal ghosts can be rapidly screened for the presence of rare anti-hemoglobin S and anti-hemoglobin C binding ghosts. In reconstruction experiments using mixtures of AS and AA cells and anti-hemoglobin S, AS ghosts as rare as 3 X 10(-5) were quantitatively recovered. Fluorescence artifacts prevented direct enumeration of AS ghosts at lower frequencies, but a two-step flow sorting-fluorescence microscope visual scanning procedure allows semiquantitative detection of anti-hemoglobin S-labeled ghosts as low as 10(--7). This method can be used for rapidly screening blood samples from individuals of normal hemoglobin A genotype for the presence of rare anti-hemoglobin S and anti-hemoglobin C binding ghosts.

Error induction and correction by mutant and wild type T4 DNA polymerases. Kinetic error discrimination mechanisms.

The fidelity of DNA synthesis as determined by the misincorporation of the base analogue 2-aminopurine in competition with adenine has been measured as a function of deoxynucleoside triphosphate substrate concentrations using purified mutator (L56), antimutator (L141), and wild type (T4D) T4 DNA polymerases. Although the rates of both incorporation and turnover of aminopurine and adenine decrease as substrate concentrations are decreased, the ratio of turnover/polymerase activity is increased. Thus, the nuclease/polymerase ratio of each of these three DNA polymerases can be controlled. The misincorporation of aminopurine decreases with decreasing substrate concentrations such that all three enzymes approach nearly identical misincorporation frequencies at the lowest substrate concentration. The increased accuracy of DNA synthesis corresponds to conditions producing a high nuclease/polymerase ratio. The misinsertion frequency for aminopurine is independent of substrate concentrations and enzyme phenotype; therefore, the increased accuracy of DNA synthesis with decreasing substrate concentrations is shown to be a result of increased nuclease activity and not increased polymerase or nuclease specificity. The data are analyzed in terms of a kinetic model of DNA polymerase accuracy which proposes that discrimination in nucleotide insertion and removal is based on the free energy difference between matched and mismatched base pairs. A value of 1.1 kcal/mol free energy difference, delta G, between adenine: thymine and aminopurine:thymine base pairs is predicted by model analysis of the cocentration dependence of aminopurine misincorporation and removal frequencies. An independent estimate of this free energy difference based on the 6-fold higher apparent Km of T4 DNA polymerase for aminopurine compared to adenine also gives a value of 1.1 kcal/mol. It is shown that the aminopurine misinsertion frequency for an enzyme having either extremely low 3'-exonuclease activity, Escherichia coli DNA polymerase I, or no measurable exonuclease activity, calf thymus DNA polymerase alpha, is 12 to 15%, which is similar to that for the T4 polymerases and consistent with delta G approximately 1.1 kcal/mol.