Jeffrey H. Miller

Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system.

We developed highly sensitive shuttle vector systems for detection of mutations formed in human cells using autonomously replicating derivatives of Epstein-Barr virus (EBV). EBV vectors carrying the bacterial lacI gene as the target for mutation were established in human cells and later returned to Escherichia coli for rapid detection and analysis of lacI mutations. The majority of the clonal cell lines created by establishment of the lacI-EBV vector show spontaneous LacI- frequencies of less than 10(-5) and are suitable for studies of induced mutation. The ability to isolate clonal lines represents a major advantage of the EBV vectors over transiently replicating shuttle vectors (such as those derived from simian virus 40) for the study of mutation. The DNA sequence changes were determined for 61 lacI mutations induced by exposure of one of the cell lines to N-nitroso-N-methylurea. A total of 33 of 34 lacI nonsense mutations and 26 of 27 missense mutations involve G X C to A X T transitions. These data provide support for the mutational theory of cancer.

On the formation of spontaneous deletions: the importance of short sequence homologies in the generation of large deletions.

Using lacl-Z fusion strains of Escherichia coli we have devised systems that detect deletions of varying lengths. We examined deletions 700-1000 base pairs long, and genetically characterized over 250 spontaneous deletions. Of these, we analyzed 24 by direct DNA sequencing and 18 by inspection of restriction fragment patterns. Deletions of this size occur almost exclusively at short repeated sequences in both (recA+ and recA- strain backgrounds, but are detected 25-fold more frequently in a recA+ background. The frequency of deletion formation correlates with the extent of homology between the short repeated sequences, although other factors may be involved. The largest hotspot, which accounts for 60% of the deletions detected, involves the largest homology in the system (14 of 17 base pairs). Altering a single base pair within this homology reduces deletion incidence by an order of magnitude. We discuss possible mechanisms of deletion formation and consider its relationship to the excision of transposable elements.

Genetic studies of the lac repressor: I. Correlation of mutational sites with specific amino acid residues: Construction of a colinear gene-protein map

Different nonsense mutations in the lacI gene have been assigned to the exact points in the gene coding for specific amino acid residues in the lac repressor, by taking advantage of the specificity of mutagens and the knowledge of the full protein sequence of the repressor. The identifications are made by correlating the limited number of amber sites found after mutagenesis with mutT with the tyrosine residues in the repressor, and the amber and ochre sites found after 2-aminopurine treatment with the tryptophan and glutamine residues. This information, together with the fine structure deletion map reported in the accompanying paper (Schmeissner et al. , 1977), has been used to construct a combined gene-protein map, in which deletion intervals on the genetic map are correlated with known segments of the protein sequence.

Genetic studies of the lac repressor: IV. Mutagenic specificity in the lacI gene of Escherichia coli

Abstract An extensive set of amber and ochre sites in the lacI gene has been characterized with respect to the base change required to generate the nonsense codon (Miller et al. , 1977; Coulondre & Miller, 1977). These mutations have been used to analyze the forward mutational spectrum of a series of mutagens in Escherichia coli . The sites induced by N′ -methyl- N′ -nitro- N -nitrosoguanidine, ethyl methanesulfonate, 4-nitroquinoline-1-oxide, and ultraviolet light, were examined, as well as those which arose spontaneously. Sites induced by the G · C → A · T transition were compared with those generated by 2-aminopurine mutagenesis. All together, more than 4000 independent occurrences of amber and ochre mutations were tabulated in order to define the respective mutagenic specificities. With the exception of the A · T → G · C change, all base substitutions lead to the generation of nonsense codons from wild-type. The A · T → G · C transition was monitored in a reversion system, in which the ochre to amber conversion (UAA → UAG) was scored, as well as the UAA → CAA reversion. Both NG ‡ and EMS were found to be highly specific for the G · C → A · T transition, less than 1% transversions appearing in either case. At between 1% and 5% the level of the G · C → A · T change, NG can stimulate the A · T → G · C transition. EMS stimulates the A · T → G · C transition at a significantly lower rate. NQO is also highly specific for G · C base-pairs, but approximately 10% of the changes found at these sites are transversions. Mutations found spontaneously or after irradiation with ultraviolet light showed none of the specificities found for EMS, NG or NQO. All transversions were detected in both cases. Moreover, a significant number of tandem double base changes were found to be induced by u.v. irradiation. Some of these have been verified directly by protein sequencing. The frequencies of occurrence of amber and ochre mutations arising from the G · C → A · T transition have been compared for different mutagens, revealing several striking hotspots. The implications of these findings with respect to the mechanism of mutagenesis and the application of different mutagens are discussed.

Genetic studies of the lac repressor. VII. On the molecular nature of spontaneous hotspots in the lacI gene of Escherichia coli.

Abstract 140 independently occurring spontaneous mutations in the lacI gene of Escherichia coli have been examined genetically and physically. DNA sequence analysis of a genetic “hotspot” shows that the tandemly repeating sequence 5′-C-T-G-G-C-T-G-G-C-T-G-G-3′ generates mutations at a high rate, either deleting or adding one unit of four nucleotides (C-T-G-G). Twelve larger deletion mutations have also been sequenced; seven of these were formed by eliminating segments between repeated sequences of five or eight nucleotides, one copy of the repeated sequence remaining after the deletion. Possible mechanisms accounting for the involvement of repeated sequences in the creation of spontaneous mutations are considered.

Effects of surrounding sequence on the suppression of nonsense codons

Using a lacI-Z fusion system, we have determined the efficiency of suppression of nonsense codons in the I gene of Escherichia coli by assaying beta-galactosidase activity. We examined the efficiency of four amber suppressors acting on 42 different amber (UAG) codons at known positions in the I gene, and the efficiency of a UAG suppressor at 14 different UGA codons. The largest effects were found with the amber suppressor supE (Su2), which displayed efficiencies that varied over a 35-fold range, and with the UGA suppressor, which displayed a 170-fold variation in efficiency. Certain UGA sites were so poorly suppressed (less than 0.2%) by the UGA suppressor that they were not originally detected as nonsense mutations. Suppression efficiency can be correlated with the sequence on the 3 side of the codon being suppressed, and in many cases with the first base on the 3 side. In general, codons followed by A or G are well suppressed, and codons followed by U or C are poorly suppressed. There are exceptions, however, since codons followed by CUG or CUC are well suppressed. Models explaining the effect of the surrounding sequence on suppression efficiency are considered in the Discussion and in the accompanying paper.

Genetic studies of the lac repressor: III. Additional correlation of mutational sites with specific amino acid residues

Abstract The complete results of the analysis of over 5300 independently derived nonsense mutations in the lacI gene are presented. These have been mapped and divided into specific sites. A total of 105 nonsense mutations derived from 90 different codons can be distinguished, of which several are the result of tandem double base changes induced by ultraviolet light. With the aid of results determined in a preceding paper (Miller et al. , 1977), the majority of these mutations have been assigned to points in the gene coding for specific residues in the lac repressor. This allows a detailed correlation of the physical and genetic map. Recombination studies have been carried out using mutations at known sites. For crosses involving mutations separated by less than 30 nucleotides (the main object of this study), a significant lack of agreement between distance and recombination frequency has been found.

DNA sequence at the integration sites of the insertion element IS1

We have detected two independent occurrences of insertion mutations in the lacl gene of E. Coli, and have used small plasmids carrying the l gene to purify large amounts of DNA containing these insertions. Analyses with restriction endonucleases and DNA sequencing techniques establish that both insertions involve the previously characterized element IS1. In each case, the integration of IS1 into the l gene DNA is associated with a directly repeated sequence of 9 nucleotides appearing at each end of the insertion element. Since one of these sequences was present in the wild-type gene, the second sequence either preexisted in the IS1 before integration, or else was generated by the process of insertion itself. The 9 base repeat is different in both cases. We discuss the relevance of these findings to the mechanism of integration of transposable elements.

Genetic and sequence analysis of frameshift mutations induced by ICR-191☆

Abstract A genetic and sequence analysis of 373 ICR-191-induced mutations in the lacI gene of Escherichia coli reveals that 365 of the mutations (97·9%) are frameshifts involving the addition or deletion of a single base-pair from a sequence including, in one case, a sequence. Some of the remaining eight mutations (2·1%) represent the loss or gain of a base-pair from a sequence. Certain mutational sites are relative hotspots for ICR-191-induced mutations, and we have analyzed the role of surrounding sequences on relative mutation rates. The preference for +1 frameshifts or −1 frameshifts is site-specific, so that at some sites +1 frameshifts predominate by a 10:1 ratio, whereas at other sites −1 frameshifts are favored by an approximately 2:1 ratio. The characterized frameshift mutations in lacI described here are useful for constructing systems to detect other frameshift and deletion mutations.

Genetic studies of the lac repressor. IX. Generation of altered proteins by the suppression of nonsence mutations.

Abstract We have generated more than 300 altered lac repressor proteins carrying known amino acid replacements, by employing nonsense mutations at 90 positions in the lacI gene together with eight different nonsense suppressors. This allows the substitution of lysine, serine, tyrosine, leucine and glutamine at virtually all of the respective positions in the repressor, and tryptophan at ten positions in the repressor. Since most of the nonsense sites have been correlated with specific codons in the lacI messenger RNA, in almost all cases the position of the substituted residue is known. This process results in the creation of a large number of mutant phenotypes. We have analyzed the effects of each substitution in vivo , and in several cases studied partially purified repressors in vitro . The properties of the altered proteins have been compared with the position and nature of each exchanged residue. We discuss the implications of these findings with regard to repressor structure in particular, and to protein structure in general. Further applications of the suppression method are also considered.