We describe an assay for determining the frequency and specificity of mutations occurring at hot spots within a population of DNA molecules. The procedure consists of (a) annealing the DNA population with a labeled oligonucleotide designed to prime DNA synthesis at the mutational hot spot; (b) DNA elongation in the presence of a single dideoxynucleoside triphosphate together with 1-3 deoxynucleoside triphosphates, and (c) quantitation of all limit elongation products by high-resolution gel electrophoresis followed by autoradiography and computing densitometry. Derivation of mutational frequency and specificity over a wide range of values is demonstrated for M13 viral DNA mixtures containing defined proportions of wild-type and mutant DNAs, as well as for M13 viral DNA populations obtained by transfection of DNA bearing a defined site-specific ethenocytosine lesion. The assay is shown to yield results similar to those obtained by laborious clone-by-clone sequencing of viral progeny. The method is not affected significantly by several tested variables and appears to be suitable for use as a quantitative assay for sequence microheterogeneity at defined positions within DNA populations. Application of the methodology demonstrates that ethenocytosine, an exocyclic DNA lesion induced by carcinogens such as vinyl chloride and urethane, is a highly efficient mutagenic lesion with a mutational specificity expected for noninstructive lesions.

Mutagenic action of chemical and physical mutagens is mediated through DNA damage and subsequent misreplication at sites of unrepaired damage. Most DNA damage is noninstructive in the sense that the causative chemical modification either destroys the template information or renders it inaccessible to the DNA polymerase. Noninstructive adducts possess high genotoxicity because they stop DNA replication. Replication past noninstructive adducts is thought to depend on induced functions in addition to the regular replication machinery. In Escherichia coli, noninstructive DNA damage leads to induction of the SOS regulon, which in turn is thought to provide the inducible functions required for replicative bypass of the lesion. Because of the absence of accessible template instruction, base incorporation opposite noninstructive lesions is inherently error-prone and results in mutagenesis. Ethenocytosine (epsilon C), an exocyclic DNA lesion induced by carcinogens such as vinyl chloride and urethane, is a highly mutagenic, noninstructive lesion on the basis of its template characteristics in vivo and in vitro. However, mutagenesis at epsilon C does not require SOS functions, as evidenced by efficient mutagenesis in recA-deleted E. coli. Even though efficient mutagenesis in recA-deleted cells shows a lack of SOS dependence, the question remains whether SOS induction can modulate mutagenesis opposite epsilon C. To examine the possible contribution of SOS functions to mutagenesis at epsilon C, we constructed an M13 duplex circular DNA molecule containing an epsilon C residue at a unique site. The construct was transfected into nonirradiated or UV-irradiated E. coli.(ABSTRACT TRUNCATED AT 250 WORDS)

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Multidrug resistance genes (mdr) that encode P-glycoproteins (P-gp) are transcriptionally regulated in normal tissues and in some multidrug-resistant (MDR) cells. Several lines of evidence suggest that regulation of P-gp overexpression at the transcriptional level is also important in human tumors. In murine MDR cells, mdr1a and/or mdr1b genes are overexpressed and P-gp isoforms are overproduced. To identify the mdr1a promoter regions that are required for transcription, the promoter has been linked to the chloramphenicol acetyltransferase (CAT) gene in transient expression vectors. 5'-Deletions of the promoter sequences have demonstrated that the region between -155 to +89 bp is crucial for basal activity of the mdr1a gene. DNase I footprinting, methylation interference, and gel retardation assays identified two nuclear protein binding sites within these sequences. One of the nuclear protein binding sites contains an 11-bp DNA sequence that interacts with nuclear protein(s) and is conserved in the promoters of the murine mdr1a and mdr1b, hamster pgp1, and human MDR1 genes. The conserved SP1 site (5'-GGGCGGG-3') that is present further downstream was shown to interact with its nuclear factor. These observations suggest that at least part of mdr gene transcriptional regulation is mediated by conserved mdr cis-regulatory elements and common nuclear factors.