Two-temperature PCR and heteroduplex detection: application to rapid cystic fibrosis screening.

We describe a rapid two-temperature PCR protocol for amplification of genomic DNA applied to the region of the most common mutation (delta F508) of the cystic fibrosis gene. Amplification products are detected as homo- or heteroduplexes on polyacrylamide gels as previously described. Data using two-temperature PCR show complete concordance with allele-specific hybridization after classical three-temperature PCR in 105 normal, carrier and affected individuals. Clinical application is demonstrated in a family which was uninformative by traditional RFLP linkage analysis. Two-temperature PCR may offer advantages of speed and specificity over three-temperature PCR in many clinical and research applications.

RecA-mediated cleavage activates UmuD for mutagenesis: mechanistic relationship between transcriptional derepression and posttranslational activation.

The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli. It has been shown that the UmuD protein shares homology with LexA, the repressor of the SOS genes. In this paper we describe a series of genetic experiments that indicate that the purpose of RecA-mediated cleavage of UmuD at its bond between Cys-24 and Gly-25 is to activate UmuD for its role in mutagenesis and that the COOH-terminal fragment of UmuD is necessary and sufficient for the role of UmuD in UV mutagenesis. Other genetic experiments are presented that (i) support the hypothesis that the primary role of Ser-60 in UmuD function is to act as a nucleophile in the RecA-mediated cleavage reaction and (ii) raise the possibility that RecA has a third role in UV mutagenesis besides mediating the cleavage of LexA and UmuD.

Survival and mutagenesis of bacteriophage T7 damaged by methyl methanesulfonate and ethyl methanesulfonate.

We have examined survival and mutagenesis of bacteriophage T7 after exposure to the alkylating agents methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS). It was found that although both alkylating agents caused increased reversion of specific T7 mutations, EMS caused a higher frequency of reversion than did MMS. Exposure of the host cells to ultraviolet light so as to induce the SOS system resulted in increased survival (Weigle reactivation) of T7 phage damaged with either EMS or MMS. However, after SOS induction of the host we did not detect an accompanying increase in mutation frequency measured as either reversion of specific T7 mutants or by generation of mutations in the T7 gene that codes for phage ligase. Neither mutation frequency nor survival of alkylated phage was affected by the umuD,C mutation in the Escherichia coli host nor by the presence of plasmid pKM101. This may mean that the mode of Weigle reactivation that is detected in T7 is not mutagenic in nature.

Mutagenesis of bacteriophage T7 and T7 DNA by alkylation damage.

We have developed a new assay for in vitro mutagenesis of bacteriophage T7 DNA that measures the generation of mutations in the specific T7 gene that codes for the phage ligase. This assay was used to examine mutagenesis caused by in vitro DNA synthesis in the presence of O6-methylguanosine triphosphate. Reversion of one of the newly generated ligase mutants by ethyl methanesulfonate was also tested.

Inducible reactivation of bacteriophage T7 damaged by methyl methanesulfonate or UV light.

We examined the effects of host mutations affecting "SOS"-mediated UV light reactivation on the survival of bacteriophage T7 damaged by UV light or methyl methanesulfonate (MMS). Survival of T7 alkylated with MMS was not affected by the presence of plasmid pKM101 or by a umuC mutation in the host. The survival of UV light-irradiated T7 was similar in umuC+ and umuC strains but was slightly enhanced by the presence of pKM101. When phage survival was determined on host cells preirradiated with a single inducing dose of UV light, these same strains permitted higher survival than that seen with noninduced cells for both UV light- and MMS-damaged phage. The extent of T7 reactivation was approximately proportional to the UV light inducing dose inflicted upon each bacterial strain and was dependent upon phage DNA damage. Enhanced survival of T7 after exposure to UV light or MMS was also observed after thermal induction of a dnaB mutant. Thus, lethal lesions introduced by UV light or MMS are apparently repaired more efficiently when host cells are induced for the SOS cascade, and this inducible reactivation of T7 is umuC+ independent.

Mutagenesis of bacteriophage T7 in vitro by incorporation of O6-methylguanine during DNA synthesis.

An in vitro system in which bacteriophage T7 DNA is replicated and efficiently packaged into procapsids to form viable phage has been used to examine mutagenesis. The fidelity of replication was assayed both by measuring reversion of an amber mutation in an essential gene and by generation of temperature-sensitive mutants among the phage produced in vitro. Under standard reaction conditions, the fidelity of DNA replication is about equal to that normally found in vivo. However, when O6-methyldeoxyguanosine triphosphate is included in the reaction, O6-methylguanine is incorporated into newly synthesized DNA and the mutation frequencies increase 10- to 70-fold over the control. These experiments demonstrate in vitro mutagenesis with the T7 DNA replication-packaging system and provide more direct evidence for the premutagenic role of O6-methylguanine.

In vitro host cell reactivation of alkylated bacteriophage T7 deoxyribonucleic acid by repair-deficient strains of Escherichia coli.

An in vitro system capable of packaging bacteriophage T7 deoxyribonucleic acid (DNA) into phage heads to form viable phage particles has been used to monitor the biological consequences of DNA dam aged by alkylating agents, and an in vitro DNA replication system has been used to examine the ability of alkylated T7 DNA to serve as template for DNA synthesis. The survival of phage resulting from in vitro packaging of DNA preexposed to various concentrations of methyl methane sulfonate or ethyl methane sulfonate closely paralleled the in vivo situation, in which intact phage were exposed to the alkylating agents. Host factors responsible for survival of alkylated T7 have been examined by using wild-type strains of EScherichia coli and mutants deficient in DNA polymerase I (polA) or 3-methyladenine-DNA glycosylase (tag). For both in vivo and in vitro situations, a deficiency in 3-methyladenine-DNA glycosylase dramatically reduced phage survival relative to that in the wild type, whereas a deficiency in DNA polymerase I had an intermediate effect. Furthermore, when the tag mutant was used as an indicator strain, phage survival was enhanced when alkylated DNA was packaged with extracts prepared from a wild-type strain in place of the tag mutant or by complementing a tag extract with an uninfected tag+ extract, indicating in vitro repair during packaging.

Capacity for postreplication repair correlated with transducibility in Rec- mutants of Bacillus subtilis.

Bacillus subtilis strains deficient in transduction, transformation, or both were examined for the ability to remove pyrimidine dimers and to convert deoxyribonucleic acid newly synthesized after ultraviolet irradiation to high molecular weight. In one strain deficient in both recombination processes, short pieces of deoxyribonucleic acid synthesized after irradiation were not converted to high molecular weight. Two transformable strains deficient in transduction were also deficient in postreplication repair (i.e., joining of newly synthesized DNA fragments), whereas a nontransformable strain that was normal in transduction was proficient in postreplication repair. None of the transformable strains showed deficiencies in repair resynthesis or ligase activity. Our results suggest that some recombinational events may be common to transduction and postreplication repair but not to transformation, emphasizing the difference between these two pathways for genetic exchange.

Postreplication repair of deoxyribonucleic acid and daughter strand exchange in uvr- mutants of Bacillus subtilis.

The fate of pyrimidine dimers in deoxyribonucleic acid (DNA) newly synthesized by Bacillus subtilis after ultraviolet irradiation was monitored by use of a damage-specific endonuclease that introduces single-strand breaks adjacent to nearly all of the dimer sites. Two Uvr- strains, one defective in the initiation of dimer excision and the other defective in a function required for efficient dimer excision, were found to be similar to their wild-type parent in the kinetics and extent of converting low-molecular-weight DNA newly synthesized after ultraviolet irradiation to high molecular weight. In the Uvr- strains large molecules of newly synthesized DNA remained susceptible to nicking by the damage-specific endonuclease even after extended incubation in growth medium, whereas the enzyme-sensitive sites were rapidly removed from both preexisting and newly synthesized DNA in Uvr+ cells. Our results support the hypothesis that postreplication repair in bacteria includes recombination between dimer-containing parental DNA strands and newly synthesized strands.