Alasdair J.E. Gordon

Missense mutation in the lacI gene of Escherichia coli: Inferences on the structure of the repressor protein

The lac repressor has been studied extensively but a precise three-dimensional structure remains unknown. Studies using mutational data can complement other information and provide insight into protein structure. We have been using the lacI gene-repressor protein system to study the mutational specificity of spontaneous and induced mutation. The sequencing of over 6000 lacI- mutations has revealed 193 missense mutations generating 189 amino acid replacements at 102 different sites within the lac repressor. Replacement sites are not distributed evenly throughout the protein, but are clustered in defined regions. Almost 40% of all sites and over one-half of all substitutions found occur within the amino-terminal 59 amino acid residues, which constitute the DNA-binding domain. The core domain (residues 60 to 360) is less sensitive to amino acid replacement. Here, substitution is found in regions involved in subunit aggregation and at sites surrounding residues that are implicated in sugar-binding. The distribution and nature of missense mutational sites directs attention to particular amino acid residues and residue stretches.

Spontaneous and 9-aminoacridine-induced frameshift mutagenesis: second-site frameshift mutation within the N-terminal region of thelacI gene ofEscherichia coli

SummaryA novel forward mutational system, based on the acquisition of an Iq-d dominant phenotype from an initial Iq− recessive state, was used to identify second-site frameshift mutation [±1(±3n) events] within the N-terminal region of thelacI gene ofEscherichia coli. The DNA sequences are described of forty-six spontaneous and twenty 9-aminoacridine(9-AA)-induced second site mutations. Although −1 frameshift events dominate both spectra, the nature and site specificity of these events clearly distinguish two mutational distributions. The spontaneous distribution contains two −(A: T) frameshift hotspots; one within a monotonic A5 run (9 occurrences), the other at a 5′-CACAACAAC-3′ sequence (12 occurrences). In contrast 17 of the 20 mutations recovered after 9-AA treatment involve the loss of a G: C pair, 14 of which occur at a single site (5′-CGGGC-3′). The striking specificity of the observed mutational hotspots is of interest since this open genetic target contains similar sequences which were infrequently recovered.

Comparison of the Mutational Specificities Exhibited by BPDE in Escherichia Coli and CHO Cells

BPDE is presumably the ultimate reactive metabolite of benzo[a]pyrene (B[a]P), a well known carcinogenic environmental pollutant. It has become evident that most chemical carcinogens are active only after metabolism to an ultimate carcinogenic and mutagenic form. These ultimate carcinogens tend to be electron deficient and thus react with nuclephilic sites which are abundant in DNA. It has been established that B[a]P is metabolized by the mixed function oxygenases to a variety of products, including the four enantiomeric forms of BPDE (anti or syn; ( + ) or (−)) (Fahl, 1982). Interestingly, metabolism of B[a]P in mammalian cells produces primarily the (+) anti-BPDE isomer (Yang, et al., 1978). However, not all DNA reactions with these ultimate carcinogens are of equal biological importance. It has been shown at the hprt locus in CHO cells that the respective mutagenic efficiency of BPDE is (+)anti > >(−)anti = (+/−)syn, and remarkably the reverse is seen at the crpt locus in TA100 bacteria (−) anti = (+/−) syn > ( + ) anti (Stevens, et al., 1985). It is also known that the ( + ) anti enantiomer is more than 60 fold more active as a tumor initiator in CD-1 and Sencar mice (Pelling, et al., 1984).