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Site specifically modified plasmids were used to determine the mutagenic effects of single arylamine adducts in bacterial cells. A synthetic heptadecamer bearing a single N-(guanin-8-yl)-2-aminofluorene (AF) or N-(guanin-8-yl)-2-(acetylamino)fluorene (AAF) adduct was used to introduce the adducts into a specific site in plasmid DNA that contained a 17-base single-stranded region complementary to the modified oligonucleotide. Following transformation of bacterial cells with the adduct-bearing DNA, putative mutants were detected by colony hybridization techniques that allowed unbiased detection of all mutations at or near the site of the adduct. The site-specific AF or AAF adducts were also placed into plasmid DNA that contained uracil residues on the strand opposite that bearing the lesions. The presence of uracil in one strand of the DNA decreases the ability of the bacterial replication system to use the uracil-containing strand, thereby favoring the use of the strand bearing the adducts. In a comparison of the results obtained with site specifically modified DNA, either with or without uracil, the presence of the uracil increased the mutation frequencies of the AF adduct by greater than 7-fold to 2.9% and of the AAF adduct by greater than 12-fold to 0.75%. The mutation frequency of the AF adduct was greatly reduced in a uvrA- strain while no mutations occurred with the AAF adduct in this strain. The sequence changes resulting from these treatments were dependent on adduct structure and the presence or absence of uracil on the strand opposite the adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

The synthetic oligonucleotide heptamer 5'-ATCCGTC-3' was reacted in vitro with N-acetoxy-N-(trifluoroacetyl)-2-aminofluorene and the resulting product isolated by reverse-phase high-performance liquid chromatography (HPLC). This purified oligonucleotide, which was shown by chemical and enzymatic analysis to be a heptamer containing a single N-(deoxyguanin-8-yl)-2-aminofluorene adduct, was then used to situate the putatively mutagenic aminofluorene lesion within the genome of M13 mp9 by ligating it into a complementary single-stranded region located at a specific site in the negative strand of the duplex M13 mp9 DNA molecule. The presence of the adduct at the anticipated location was confirmed by taking advantage of the facts that AF adducts inhibit many restriction enzymes when located in or near their restriction sites and that the AF moiety should be contained within the HincII recognition sequence on M13 mp9 DNA. Upon attempted cleavage of the M13 DNA containing the site-specific AF adduct with HincII, we find that the large majority of the DNA remained circular, demonstrating the incorporation of the AF adduct in high yield into the DNA molecule at this location. This system should prove useful in vivo for the study of mutagenesis by chemical carcinogens and in vitro to study the interaction of purified DNA metabolizing proteins with a template containing a site-specific lesion.

The spin-label electron paramagnetic resonance technique has been used to compare the interactions of normal and sickle hemoglobin molecules with human erythrocyte membranes. The sickle hemoglobin molecules show an enhanced binding to membranes when compared to normal hemoglobin (HbA) molecules. Using a simple equilibrium model for hemoglobin--membrane interactions, we obtain an equilibrium dissociation constant for sickle hemoglobin of about half that of HbA at pH 7.4 in 5 mM phosphate at 20 degrees C. The interactions are very low affinity in nature and are stronger at lower pH than at pH 7.4 (Fung, 1981a). The difference between normal and sickle hemoglobin persists at both high (pH 7.4) and low (pH 6.7) pH values. The concentrations of hemoglobin at the saturation level are close to physiological concentrations. Removal of spectrin--actin protein molecules from the membranes causes little change in the interactions, indicating that the remaining membrane proteins play the primary role in hemoglobin--membrane interactions. This observation is further supported by data of spectrin--actin-depleted inside-out vesicle samples. The stronger interaction of sickle hemoglobin than normal hemoglobin with membranes is discussed in relation to the formation of irreversibly sickled cells.