SummaryThis work provides evidence that, during transcription, the mutability (propensity to mutate) of a base in a DNA secondary structure depends both on the stability of the structure and on the extent to which the base is unpaired. Zuker ' s DNA folding computer program reveals the most probable stem-loop structures (SLSs) and negative energies of folding (-∆ ∆ ∆ ∆ G) for any given nucleotide sequence. We developed an interfac-

Escherichia coli auxotrophs of leuB and argH were examined to determine if higher rates of transcription in derepressed genes were correlated with increased reversion rates. Rates of leuB and argH mRNA synthesis were determined using half-lives and concentrations, during exponential growth and at several time points during 30 min of amino acid starvation. Changes in mRNA concentration were primarily due to increased mRNA synthesis and not to increased stability. Four strains of E. coli amino acid auxotrophs, isogenic except for relA and argR, were examined. In both the leuB and argH genes, rates of transcription and mutation were compared. In general, strains able to activate transcription with guanosine tetraphosphate (ppGpp) had higher rates of mRNA synthesis and mutation than those lacking ppGpp (relA2 mutants). argR knockout strains were constructed in relA + and relA mutant strains, and rates of both argH reversion and mRNA synthesis were significantly higher in the argR knockouts than in the regulated strains. A statistically significant linear correlation between increased rates of transcription and mutation was found for data from both genes. In general, changes in mRNA half-lives were less than threefold, whereas changes in rates of mRNA synthesis were often two orders of magnitude. The results suggest that specific starvation conditions target the biosynthetic genes for derepression and increased rates of transcription and mutation.

Rochalimaea quintana is the only member of the family Rickettsiaceae that can be grown in vitro. Because of its relationship to the other members of this family, techniques developed to transform R. quintana might be applicable to the obligate intracellular bacteria of the Rickettsiaceae. These procedures are critical to understanding mechanisms of pathogenesis and the nature of obligate intracellular growth. A transformation procedure for R. quintana has been established by using electroporation techniques. Several cosmids or plasmids with replicons RK2 and RSF1010 have been successfully used to transform this organism. Transformants were obtained by selection for antibiotic resistance to chloramphenicol or kanamycin. Plasmid retention and replication has been verified by Southern blot analysis and chloramphenicol acetyltransferase assay. Experimentation with different voltage field strengths and pulse times indicate that 12.5 kV/cm at 10 ms (25 ,LF and 400 fQ) was optimal, giving a transformation frequency of -0.3% and 3 x 105 transformants per ,ug of DNA.The family Rickettsiaceae contains a number of important human pathogens that are associated with arthropods. The members of this family differ markedly and have been separated into three genera: Rickettsia, Coxiella, and Rochalimaea (29).' Both Rochalimaea quintana and Rochalimaea vinsonii can be cultivated axenically and appear to grow in epicellular locations within their eucaryotic hosts (8,27). Members of this genus appear to be more closely related to some species of the genus Rickettsia than to Coxiella burnetii. Indeed, C. burnetii does not appear to be related to either Rickettsia or Rochalimaea species (24, 25). R. quintana has been shown to have metabolic and nutritional properties similar to Rickettsia spp. (27). In support of these findings, studies of the genetic relatedness of Rochalimaea and Rickettsia species by DNA-DNA hybridization analyses reveal a small but significant degree of hybridization (16). Recently, genetic analyses utilizing highly conserved rRNA sequences have confirmed the relationship between these two genera (26). Classical genetic studies of the genera Rickettsia and Coxiella have been severely restricted because of difficulties in manipulating these obligate intracellular organisms. The ability to transform these organisms would allow the introduction of specific mutations or genes and transformants could be examined for altered biological properties in vivo. It

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Transcription drives supercoiling which forms and stabilizes single-stranded (ss) DNA secondary structures with loops exposing G and C bases that are intrinsically mutable and vulnerable to non-enzymatic hydrolytic reactions. Since many studies in prokaryotes have shown direct correlations between the frequencies of transcription and mutation, we conducted in silico analyses using the computer program, mfg, which simulates transcription and predicts the location of known mutable bases in loops of high-stability secondary structures. Mfg analyses of the p53 tumor suppressor gene predicted the location of mutable bases and mutation frequencies correlated with the extent to which these mutable bases were exposed in secondary structures. In vitro analyses have now confirmed that the 12 most mutable bases in p53 are in fact located in predicted ssDNA loops of these structures. Data show that genotoxins have two independent effects on mutagenesis and the incidence of cancer: Firstly, they activate p53 transcription, which increases the number of exposed mutable bases and also increases mutation frequency. Secondly, genotoxins increase the frequency of G-to-T transversions resulting in a decrease in G-to-A and C mutations. This precise compensatory shift in the 'fate' of G mutations has no impact on mutation frequency. Moreover, it is consistent with our proposed mechanism of mutagenesis in which the frequency of G exposure in ssDNA via transcription is rate limiting for mutation frequency in vivo.