Search

Mutator cells that lack the mismatch repair system (MMR(-)) occur at rates of 10(-5) or less in laboratory populations started from wild-type cells. We show that after selection for recombinants in an interspecies mating between Salmonella enterica serovar Typhimurium and Escherichia coli, the percentage of MMR(-) cells rises to several percent of the recombinant population, and after a second successive mating and selection, greater than 95% of the recombinants are MMR(-). Coupling a single cross and selection with either mutagenesis or selection for spontaneous mutants also results in a dramatic increase in MMR(-) cells. We discuss how horizontal transfer can result in mutator strains during adaptive evolution.

Background: Not all of the enzymatic pathways involved in genetic rearrangements have been elucidated. While some rearrangements occur by recombination at areas of high homology, others are mediated by short, often interrupted homologies. We have previously constructed an Escherichia coli strain that allows us to examine inversions at microhomologies, and have shown that inversions can occur at short inverted repeats in a recB,C-dependent fashion., Results: Here, we report on the use of this strain to define genetic loci involved in limiting rearrangements on an F' plasmid carrying the lac genes. Employing mini-Tn10 derivatives to generate insertions near or into genes of interest, we detected three loci (rmuA,B,C) that, when mutated, increase inversions. We have mapped, cloned and sequenced these mutator loci. In one case, inactivation of the sbcC gene leads to an increase in rearrangements, and in another, insertions near the recE gene lead to an even larger increase. The third gene involved in limiting inversions, rmuC, has been mapped at 86 min on the E. coli chromosome and encodes a protein of unknown function with a limited homology to myosins, and some of the SMC (structural maintenance of chromosomes) proteins., Conclusions: This work presents the first example of an anti-mutator role of the sbcC,D genes, and defines a new gene (rmuC) involved in DNA recombination.

We have examined the composition of members of mutator populations of Escherichia coli by employing an extensive set of phenotypic screens that allow us to monitor the function of >700 genes, constituting approximately 15% of the genome. We looked at mismatch repair deficient cells after repeated cycles of single colony isolation on rich medium to generate lineages that are forced through severe bottlenecks, and compared the results to those for wild-type strains. The mutator lineages continued to accumulate mutations rapidly with each increasing cycle of colony isolation. By the end of the 40th cycle, after approximately 1000 generations, most of the lineages had reduced colony size, 4% had died out, 55% had auxotrophic requirements (increasing to 80% after 60 cycles), and 70% had defects in at least one sugar or catabolic pathway. In addition, 33% had a defect in cell motility, and 26% were either temperature-sensitive or cold-sensitive lethals. On the other hand, only 3% of the wild-type lineages had detectable mutations of any type after 40 cycles. By the 60th cycle, the typical mutator cell carried 4-5 inactive genes among the 15% of the genome being monitored, indicating that the average cell carried at least 24-30 inactivated genes distributed throughout the genome. Remarkably, 30% of the lineages had lost the ability to utilize xylose as a carbon source. DNA sequencing revealed that most of the Xyl(-) mutants had a frameshift in a run of eight G's (GGGGGGGG) in the xylB gene, either adding or deleting one -G-. Further analysis indicated that rendering E. coli deficient in mismatch repair unmasks hypermutable sites in certain genes or intergenic regions. Growth curves and competition tests on lineages that passed through 90 cycles of single colony isolation showed that all lineages suffered reduced fitness. We discuss these results in terms of the value of mutators in cellular evolution.

We have constructed strains that allow a direct selection for mutators of Escherichia coli on a single plate medium. The plate selection is based on using two different markers whose reversion is enhanced by a given mutator. Plates containing limiting amounts of each respective nutrient allow the growth of ghost colonies or microcolonies that give rise to full-size colonies only if a reversion event occurs. Because two successive mutational events are required, mutator cells are favored to generate full-size colonies. Reversion of a third marker allows direct visualization of the mutator phenotype by the large number of blue papillae in the full-size colonies. We also describe plate selections involving three successive nutrient markers followed by a fourth papillation step. Different frameshift or base substitution mutations are used to select for mismatch-repair-defective strains (mutHLS and uvrD). We can detect and monitor mutator cells arising spontaneously, at frequencies lower than 10(-5) in the population. Also, we can measure a mutator cascade, in which one type of mutator (mutT) generates a second mutator (mutHLS) that then allows stepwise frameshift mutations. We discuss the relevance of mutators arising on a single medium as a result of cells overcoming successive growth barriers to the development and progression of cancerous tumors, some of which are mutator cell lines.

We previously described Escherichia coli mutator tRNAs that insert glycine in place of aspartic acid and postulated that the elevated mutation rate results from generating a mutator polymerase. We suggested that the proofreading subunit of polymerase III, epsilon, is a likely target for the aspartic acid-to-glycine change that leads to a lowered fidelity of replication, since the altered epsilon subunits resulting from this substitution (approximately 1% of the time) are sufficient to create a mutator effect, based on several observations of mutD alleles. In the present work, we extended the study of specific mutD alleles and constructed 16 altered mutD genes by replacing each aspartic acid codon, in series, with a glycine codon in the dnaQ gene that encodes epsilon. We show that three of these genes confer a strong mutator effect. We have also looked for new mutator tRNAs and have found one: a glycine tRNA that inserts glycine at histidine codons. We then replaced each of the seven histidine codons in the mutD gene with glycine codons and found that in two cases, a strong mutator phenotype results. These findings are consistent with the epsilon subunit playing a major role in the mutator effect of misreading tRNAs.

A Lac- strain of Escherichia coli that reverts by the addition of a G to a G-G-G-G-G-G sequence was used to study the proliferation of mutators in a bacterial culture. Selection for the Lac+ phenotype, which is greatly stimulated in mismatch repair-deficient strains, results in an increase in the percentage of mutators in the selected population from less than 1 per 100,000 cells to 1 per 200 cells. All the mutators detected were deficient in the mismatch repair system. Mutagenesis results in a similar increase in the percentage of mutators. Mutagenesis combined with a single selection can result in a population of more than 50% mutators when a sample of several thousand cells is grown out and selected. Mutagenesis combined with two or more successive selections can generate a population that is 100% mutator. These experiments are discussed in relation to ideas that an early step in carcinogenesis is the creation of a mutator phenotype.

Mutations in the lacZ gene were constructed that allowed the detection of mutators that caused specific transversion mutations. This allowed the detection of two new mutator genes, mut Y and mut M, which are involved in the repair of the 8-oxodguanine (8oxodG) lesion.

The MutY protein of Escherichia coli removes mismatched deoxyadenine residues from DNA. In this study, duplex oligodeoxynucleotides containing modified bases are used as model substrates for this enzyme. In contrast to a recent report [Lu, A.-L., et al. (1995) J. Biol. Chem. 270, 23582], dA:8-oxo-dG appears to be the preferred natural substrate for MutY, as evidenced by the specificity constants (kcat/Km) for dA:8-oxo-dG and dA:dG of 39 600 x 10(-6) and 383 x 10(-6) (min-1 nM-1), respectively. kcat for the duplex containing dA:dG was highest at lower pH; the rate of cleavage for the duplex containing dA:8-oxo-dG was unaffected over a pH range of 5.5-8.0. The presence of an 8-oxo function in dG increased significantly the rate of removal of dA from all substrates tested. Replacement of dA by rA reduced the specificity constant of dA:8-oxo-dG to 294 x 10(-6) (min-1 nM-1), whereas replacement of dA by 2'-O-methyladenosine virtually abolished enzymatic activity. Modifications of the dG moiety generally were better tolerated than those of dA; however, introduction of a methyl ether at the 6 position of dG produced a noncleavable substrate and replacement of dG by 2'-O-methylguanosine generated a substrate with a low specificity constant. Rates of cleavage of duplexes containing dA:dC and dA:tetrahydrofuran were three orders of magnitude lower than the reference substrate. Duplexes containing a carbocyclic analog of dA were not cleaved. A model is proposed to explain the recognition of DNA substrates by MutY and the catalytic properties of this enzyme.

We have previously described the mutator alleles mutA and mutC, which map at 95 minutes and 42 minutes, respectively, on the Escherichia coli genetic map and which stimulate transversions; the A.T-->T.A and G.C-->T.A substitutions are the most prominent. In this study we show that both mutA and mutC result from changes in the anticodon in one of four copies of the same glycine tRNA, at either the glyV or the glyW locus. This change results in a tRNA that inserts glycine at aspartic acid codons. In view of previous studies of missense suppressor tRNAs, the mistranslation of aspartic acid codons is assumed to occur at approximately 1-2%. We postulate that the mutator tRNA effect is exerted by generating a mutator polymerase and suggest that the epsilon subunit of DNA polymerase, which provides a proofreading function, is the most likely target. The implications of these findings for the contribution of mistranslation to observed spontaneous mutation rates in wild-type strains, as well as other cellular phenomena such as aging, are discussed.

Amber mutations have been constructed at 328 positions, corresponding to residues 2 to 329 in the E. coli lac repressor protein. Synthetic and naturally occurring nonsense suppressors have been used to insert, in series, 12-13 amino acids at positions specified by an amber (UAG) codon in the lacI mRNA. The resulting set of over 4000 single amino acid replacements in the lac repressor protein allows a detailed analysis of its substitution tolerance along the linear array of residues, and reveals structure-function relationships in lac repressor and in proteins in general. (1) There are two main regions in the repressor which are extremely sensitive to amino acid replacements. One, the amino-terminal 59 residues, has been implicated in DNA and operator binding by a large body of work. The second, extending from approximately residues 239 to 289/292, forms the repressor core and shares the most homology with other repressor and DNA binding proteins. (2) Throughout the rest of the protein, segments of 6 to 14 amino acids, which are highly tolerant to single amino acid replacements, appear to act as "spacers" between one or several hydrophobic residues that are relatively intolerant to substitutions. (3) We have replaced the amino acids in these tolerant regions with spans of alanine residues, from 5 to 13 amino acids. In all five of the regions tested, alanine replacements, sometimes of up to 8 amino acids, still allowed functional repressor, while deletion of the same residues destroyed repressor function. This reinforces the view that many regions of a protein do not require a specific sequence to serve as spacers between more important residues. (4) A distinct pattern of substitutions leading to the I(s) phenotype suggests the location of residues involved in inducer binding. (5) A number of general substitution patterns can be recognized. For instance, proline is not tolerated at over 40 sites which tolerate all the other amino acid replacements. Another set of sites tolerates only non-polar amino acids, whereas a third set tolerates a subset of the smallest amino acids, (serine, alanine, glycine and cysteine, and sometimes threonine and valine). (5) Overall, 93 of 328 sites (28%) tolerate all 13 amino acids tested, and 144 of 328 (44%) tolerate 12/13 or all 13 substitutions. We judge that 192 of 328 sites (59%) are generally tolerant to substitutions.

Fpg protein of Escherichia coli cleaves duplex DNA containing the oxidatively damaged base 8-oxo-7,8-dihydroguanine (Tchou, J., Kasai, H., Shibutani, S., Chung, M.-H., Laval, J., Grollman, A. P., and Nishimura, S. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4690-4694). This DNA repair enzyme contains one zinc atom/protein molecule (Boiteux, S., O'Connor, T. R., Lederer, F., Gougette, A., and Laval, J. (1990) J. Biol. Chem. 265, 3916-3922); its N-glycosylase and apurinic/apyrimidinic lyase activities are physically associated. Amino acid sequence analysis reveals a putative single zinc finger motif of the CC/CC type located near the carboxyl terminus. A gel mobility shift assay was used to assay binding of Fpg protein to a noncleavable substrate analog, namely an oligodeoxynucleotide duplex containing a single tetrahydrofuran residue. High resolution hydroxyl radical DNA footprinting showed protection centered around the tetrahydrofuran residue. No footprint was observed on the complementary strand. To establish the role of COOH-terminal zinc finger in DNA binding and/or DNA cleavage, amino acid substitutions and an amber mutation were introduced at Cys-244 (C244S, C244H, C244A, and C244amber). In addition, a double amino acid substitution was generated at Cys-244 and Cys-247 (C244S/C247S). These mutant Fpg proteins lack DNA binding or cleavage activity, as tested in crude lysates of Escherichia coli. Wild type Fpg protein contains one zinc/protein molecule, whereas the mutant Fpg protein (C244S/C247S) lacks zinc, as measured by atomic absorption spectroscopy. This mutation did not significantly alter secondary structure, as assessed by circular dichroism spectroscopy. Our results suggest that Fpg protein utilizes its single COOH-terminal zinc finger motif in DNA binding.

Using site-specific mutagenesis in vitro, we have constructed Escherichia coli strains that allow the detection of the inversion of an 800-bp segment in the lac region. The invertible segment is bounded by inverted repeats of either 12 or 23 bp. Inversions occurring at these inverted repeats will restore the Lac+ phenotype. Inversions can be detected at both short homologies at frequencies ranging from 0.5 x 10(-8) to 1 x 10(-7). These events, which have been verified by DNA sequence analysis, are reduced up to 1000-fold in strains deficient for either RecA, RecB or RecC. They are not reduced in strains deficient in the RecF, J pathway. These results show that the RecB,C,D system can mediate rearrangements at short sequence repeats, and probably plays a major role in cellular rearrangements.

Amino acid substitution analysis within a highly conserved region of Escherichia coli thymidylate synthase (TS), using suppression of amber mutations by tRNA suppressors, has yielded a bank of 124 new mutationally altered TS proteins. These mutant proteins have been used to study the structure-function relationship of the Escherichia coli TS protein at the N-terminus corresponding to residues 20 through 35. This region contains a block of amino acids whose sequence has been well conserved among other known TS proteins from various organisms. Positions 20 through 25 contain a surface loop structure and positions 26 through 35 encompass a beta-strand. We find that residues surrounding a beta-bulge structure within the beta-strand are particularly sensitive to amino acid substitution, suggesting that this structure is maintained by a highly ordered packing arrangement. Three residues in the surface loop that are present at the base of the substrate binding pocket are also sensitive to amino acid substitution. The remainder of the conserved sites, including those at the dimer interface, are tolerant to most, if not all, of the substitutions tested.

It has been previously shown both in vivo and in vitro that DNA synthesis past an oxidatively damaged form of guanine, 7,8-dihydro-8-oxoguanine (8-oxoG), can result in the misincorporation of adenine (A) opposite the 8-oxodG. In this study we show that MutY glycosylase is active on a site-specific, oxidatively damaged A/8-oxoG mispair and that it removes the undamaged adenine from this mispair. Strains that lack active MutY protein have elevated rates of G.C----T.A transversions. We find that the mutator phenotype of a mutY strain can be fully complemented by overexpressing MutM protein (Fpg protein) from a plasmid clone. The MutM protein removes 8-oxoG lesions from DNA. In addition, we have isolated a strain with a chromosomal mutation that suppresses the mutY phenotype and found that this suppressor also overexpresses MutM. Finally, a mutY mutM double mutant has a 25- to 75-fold higher mutation rate than either mutator alone. The data strongly suggest that MutY is part of an intricate repair system directed against 8-oxoG lesions in nucleic acids and that the primary function of MutY in vivo is the removal of adenines that are misincorporated opposite 8-oxoG lesions during DNA synthesis.

We have cloned and sequenced the mutR gene from Escherichia coli, which results in an increased frequency of spontaneous deletions, by using a strain carrying a Tn10 derivative inserted into mutR. The analysis of 1,286 bp of mutR sequence shows that this gene is identical to the topB gene, which encodes topoisomerase III. The increased deletion formation is the first reported phenotype for cells lacking topoisomerase III, and this suggests that topoisomerase III is involved in reactions that normally reduce the levels of spontaneous deletions.

We have cloned chromosomal DNA bordering an insert that inactivates mutM. Sequencing of this clone has revealed that the insertion element is located between the promoter and structural gene for formamidopyrimidine-DNA glycosylase (Fapy-DNA glycosylase). An overproducing clone of Fapy-DNA glycosylase complements the original mutM strain that had been isolated after EMS mutagenesis. Thus, we conclude that MutM is actually Fapy-DNA glycosylase. mutM has previously been characterized as a mutator strain that leads specifically to G.C----T.A transversions. This in vivo characterization correlates well with the mutagenic potential of one of the lesions Fapy-DNA glycosylase removes, 8-oxo-7,8-dihydro-2'-deoxyguanine (8-OxodG).

Using site-specific mutagenesis in vitro we constructed a genetic system to detect mutants with altered rates of deletion formation between short repeated sequences in Escherichia coli. After in vivo mutagenesis with chemical mutagens and transposons, the system allowed the identification of mutants with either increased or decreased deletion frequencies. One mutational locus, termed mutR, that results in an increase in deletion formation, was studied in detail. The mutR gene maps at 38.5 min on the E. coli genetic map. Since the precise excision of many transposable elements is also mediated at short repeated sequences, we investigated the effects of the mutant alleles, as well as recA, on precise excision of the transposon Tn9. Neither mutR nor recA affect precise excision of the transposon Tn9, from three different insertions in lacI, whereas these alleles do affect other spontaneous deletions in the same system. These results indicate that deletion events leading to precise excision occur principally via a different pathway than other random spontaneous deletions. It is suggested that, whereas precise excision occurs predominantly via a pathway involving replication enzymes (for instance template strand slippage), deletions on an F'factor are stimulated by recombination enzymes.

The use of suppressed nonsense mutations to generate altered proteins can greatly simplify studies in which a large number of defined mutant proteins are sought. If site-directed mutagenesis is used to generate specific mutations, than for every amber (UAG) mutation constructed, as many as 13 different amino acids can be inserted at the corresponding site in the protein. This allows a rapid screening of many altered proteins for those with interesting properties. Once identified, the interesting substitutions can be regenerated by missense changes, to avoid some of the potential problems of the method. Nonsense suppression has been used to generate more than 3300 amino acid replacements in the E. coli lac repressor, and close to 250 amino acid substitutions in E. coli thymidylate synthase.

Two transversion-specific mutator loci, mutA and mutC, were identified in Escherichia coli. Mutators with high rates of A.T----T.A transversions were identified using a screening technique that relied upon the reversion of an altered lacZ gene back to wild-type via a specific A.T----T.A transversion. Among the mutators collected, one class mapped to a previously unidentified locus that we designate mutA. Analysis of reverse mutations in lacZ and forward nonsense mutations in lacI showed that the mutA strain has higher levels of A.T----T.A and G.C----T.A transversions, and to a lesser degree A.T----C.G transversions. The mutA locus maps very near to, but is separable from, mutL, at about 95 min on the E. coli chromosome. Both its mutagenic specificity and complementation experiments confirmed that mutA is distinct from mutL and from a nearby mutator locus, miaA. The phenotype of a mutA mutL double mutator strain suggests that the mutA gene product prevents some replication errors. Another mutator, designated mutC, maps very near uvrC, at 42 min, but is distinguishable from uvrC, which has no mutator effects. The specificity of reversion of lacZ mutations in a mutC strain is identical to that in a mutA strain. Also, the behavior of a mutC mutS double mutant is identical to that of a mutA mutL double mutant. It is likely that mutA and mutC are components of the same error-avoidance system.

The mutY gene of Escherichia coli, which codes for an adenine glycosylase that excises the adenine of a G-A mispair, has been cloned and sequenced. The mutY gene codes for a protein of 350 amino acids (Mr = 39,123) and the clone genetically complements the mutY strain. The protein shows significant sequence homology to E. coli endonuclease III, an enzyme that has previously been shown to have glycosylase activity on damaged base pairs. Sequence analysis suggests that, like endonuclease III, MutY is an iron-sulfur protein with a [4Fe-4S]2+ cluster.

Using synthetic oligonucleotides, we have constructed a collection of Escherichia coli amber suppressor tRNA genes. In order to determine their specificities, these tRNAs were each used to suppress an amber (UAG) nonsense mutation in the E. coli dihydrofolate reductase gene fol. The mutant proteins were purified and subjected to N-terminal sequence analysis to determine which amino acid had been inserted by the suppressor tRNAs at the position of the amber codon. The suppressors can be classified into three groups on the basis of the protein sequence information. Class I suppressors, tRNA(CUAAla2), tRNA(CUAGly1), tRNA(CUAHisA), tRNA(CUALys) and tRNA(CUAProH), inserted the predicted amino acid. The class II suppressors, tRNA(CUAGluA), tRNA(CUAGly2) and tRNA(CUAIle1) were either partially or predominantly mischarged by the glutamine aminoacyl tRNA synthetase. The class III suppressors, tRNA(CUAArg), tRNA(CUAAspM), tRNA(CUAIle2), tRNA(CUAThr2), tRNA(CUAMet(m)) and tRNA(CUAVal) inserted predominantly lysine.

Using synthetic oligonucleotides, we have constructed 17 tRNA suppressor genes from Escherichia coli representing 13 species of tRNA. We have measured the levels of in vivo suppression resulting from introducing each tRNA gene into E. coli via a plasmid vector. The suppressors function at varying efficiencies. Some synthetic suppressors fail to yield detectable levels of suppression, whereas others insert amino acids with greater than 70% efficiency. Results reported in the accompanying paper demonstrate that some of these suppressors insert the original cognate amino acid, whereas others do not. We have altered some of the synthetic tRNA genes in order to improve the suppressor efficiency of the resulting tRNAs. Both tRNA(CUAHis) and tRNA(CUAGlu) were altered by single base changes, which generated -A-A- following the anticodon, resulting in a markedly improved efficiency of suppression. The tRNA(CUAPro) was inactive, but a hybrid suppressor tRNA consisting of the tRNA(CUAPhe) anticodon stem and loop together with the remainder of the tRNA(Pro) proved highly efficient at suppressing nonsense codons. Protein chemistry results reported in the accompanying paper show that the altered tRNA(CUAHis) and the hybrid tRNA(CUAPro) insert only histidine and proline, respectively, whereas the altered tRNA(CUAGlu) inserts principally glutamic acid but some glutamine. Also, a strain deficient in release factor I was employed to increase the efficiency of weak nonsense suppressors.

We have used site-directed mutagenesis to alter bases in lacZ near the region encoding essential residues in the active site of beta-galactosidase. The altered sequences generate runs of six or seven identical base pairs which create a frameshift, resulting in a Lac- phenotype. Reversion to Lac+ in each strain can occur only by a specific frameshift at these sequences. Monotonous runs of A's (or of T's on the opposite strand) and G's (or C's) have been constructed, as has an alternating -C-G- sequence. These specific frameshift indicator strains complement a set of six previously described strains which detect each of the base substitutions. We have examined a variety of mutagens and mutators for their ability to cause reversion to Lac+. Surprisingly, frameshifts are well stimulated at many of these runs by ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and 2-amino-purine, mutagens not widely known to induce frameshifts. A comparison of ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and 2-aminopurine frameshift specificity with that found with a mutH strain suggests that these mutagens partially or fully saturate or inactivate the methylation-directed mismatch repair system and allow replication errors leading to frameshifts to escape repair. This results in a form of indirect mutagenesis, which can be detected at certain sites.

By using site-directed oligonucleotide mutagenesis, amber nonsense stop codons (5'-TAG-3') have been introduced at 20 sites in the Escherichia coli thymidylate synthase gene. By transforming the thyA mutant plasmids into 13 strains, each of which harbor different amber suppressor tRNAs, we were able to generate over 245 amino acid substitutions in E. coli thymidylate synthase (EC 2.1.1.45). Growth characteristics of these mutants have been studied, yielding a body of information that includes some surprising results in light of the recently published crystal structure of the enzyme.

Site-directed substitutions (Asp, Gly, Gln, His, and Lys) were made for Glu-461 of beta-galactosidase (Escherichia coli). All substitutions resulted in loss of most activity. Substrates and a substrate analog inhibitor were bound better by the Asp-substituted enzyme than by the normal enzyme, about the same for enzyme substituted with Gly, but only poorly when Gln, His, or Lys was substituted. This shows that Glu-461 is involved in substrate binding. Binding of the positively charged transition state analog 2-aminogalactose was very much reduced with Gly, Gln, His, and Lys, whereas the Asp-substituted enzyme bound this inhibitor even better than did the wild-type enzyme. Since Asp, like Glu, is negatively charged, this strongly supports the proposal that one role of Glu-461 is to electrostatically interact with a positively charged galactosyl transition state intermediate. The substitutions also affected the ability of the enzyme to bind L-ribose, a planar analog of D-galactose that strongly inhibits beta-galactosidase activity. This indicates that the binding of a planar "galactose-like" compound is somehow mediated through Glu-461. The data indicated that the presence of Glu-461 is highly important for the acid catalytic component of kappa 2 (glycosylic bond cleavage or "galactosylation"), and therefore Glu-461 must be involved in a concerted acid catalytic reaction, presumably by stabilizing a developing carbonium ion. The kappa 2 values with o- and p-nitrophenyl-beta-D-galactopyranoside as substrates varied more or less as did the K8 values, indicating that most of the glycolytic bond breaking activity found for the enzymes from the mutants with these substrates was probably a result of strain or other such effects. The kappa 3 values (hydrolysis or "degalactosylation") of the substituted enzymes were also low, indicating that Glu-461 is important for that part of the catalysis. The enzyme with His substituted for Glu-461 had the highest kappa 3 value. This is probably a result of the formation of a covalent bond between His and the galactosyl part of the substrate.

Amber suppressor genes corresponding to Escherichia coli tRNAPhe and tRNACys have been constructed for use in amino acid substitution studies as well as protein engineering. The genes for either tRNAPheGAA or tRNACysGCA both with the anticodon 5' CTA 3' were assembled from four to six oligonucleotides, which were annealed and ligated into a vector. The suppressor genes are expressed constitutively from a synthetic promoter, derived from the promoter sequence of the E. coli lipoprotein gene. The tRNAPhe suppressor (tRNAPheCUA) is 54-100% efficient in vivo, while the tRNACys suppressor (tRNACysCUA) is 17-50% efficient. To verify that the suppressors insert the predicted amino acids, both genes were used to suppress an amber mutation in a protein coding sequence. NH2-terminal sequence analysis of the resultant proteins revealed that tRNAPheCUA and tRNACysCUA insert phenylalanine and cysteine, respectively. To demonstrate the potential of these suppressors, tRNAPheCUA and tRNACysCUA have been used to effect amino acid substitutions at specific sites in the E. coli lac repressor.

Forty-nine amino acid substitutions were made at four positions in the Escherichia coli enzyme beta-galactosidase; three of the four targeted amino acids are thought to be part of the active site. Many of the substitutions were made by converting the appropriate codon in lacZ to an amber codon, and using one of 12 suppressor strains to introduce the replacement amino acid. Glu-461 and Tyr-503 were replaced, independently, with 13 amino acids. All 26 of the strains containing mutant enzymes are Lac-. Enzyme activity is reduced to less than 10% of wild type by substitutions at Glu-461 and to less than 1% of wild type by substitutions at Tyr-503. Many of the mutant enzymes have less than 0.1% wild-type activity. His-464 and Met-3 were replaced with 11 and 12 amino acids, respectively. Strains containing any one of these mutant proteins are Lac+. The results support previous evidence that Glu-461 and Tyr-503 are essential for catalysis, and suggest that His-464 is not part of the active site. Site-directed mutagenesis was facilitated by construction of an f1 bacteriophage containing the complete lacZ gene on a single EcoRI fragment.

A temperature-sensitive mutant having a lethal mutation in the gene for the beta subunit of RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) exhibits an apparent 2- to 3-fold decrease in the rates of both beta and beta' subunit synthesis at the non-permissive temperature, relative to total protein. In contrast, a temperature-sensitive mutant with a lethal mutation in the gene encoding beta' has a 5- to 6-fold increase in the rates of beta and beta' synthesis at 42 degrees. These beta and beta' mutants also exhibit rapid degradation of both subunits at the high temperature.

The DNA binding fragment from Escherichia coli lac repressor, the N-terminal 56 amino acid residue "headpiece", has been examined by high-resolution 1H NMR spectroscopy at 360 MHz. The aromatic region has been examined in detail along with the four headpieces of altered repressors that are each missing one of the tyrosines, respectively. The spectra here show more resolved resonances and correct errors in the resonance assignments that have been published by Ribeiro et al. (1981b) Ribeiro, A. A., Wemmer, D., Bray, R. P., Wade-Jardetzky, N. G., & Jardetzky, O. (1981) Biochemistry 20, 818-823]. These corrections allow an interpretation of the spectroscopic observations that is now consistent with the extensive genetic analysis that has been carried out with the lac repressor gene. In addition, nuclear Overhauser enhancement measurements give a guide to the interresidue distances among the aromatic residues in this protein fragment.

We describe the construction of six strains of Escherichia coli with different mutations at the same coding position in the lacZ gene, which specifies the active site glutamic acid residue at position 461 in beta'-galactosidase. Each strain is Lac- and reverts to Lac+ only by restoring the glutamic acid codon. The strains have been designed so that each reverts via one of the six base substitutions. The set of strains allows detection of each transition and transversion simply by monitoring the Lac- to Lac+ frequency, as demonstrated here with characterized mutagens and mutator alleles. These strains are useful for rapidly determining the mutagenic specificity of mutagens at a single site, for detecting low levels of stimulation of certain base substitutions, for monitoring specific base changes in response to various experimental conditions or strain backgrounds, and for isolating new mutator strains.

We have used a strain with an altered lacZ gene, which reverts to wild type via only certain transversions, to detect transversion-specific mutators in Escherichia coli. Detection relied on a papillation technique that uses a combination of beta-galactosides to reveal blue Lac+ papillae. One class of mutators is specific for the G.C----T.A transversion as determined by the reversion pattern of a set of lacZ mutations and by the distribution of forward nonsense mutations in the lacI gene. The locus responsible for the mutator phenotype is designated mutY and maps near 64 min on the genetic map of E. coli. The mutY locus may act in a similar but reciprocal fashion to the previously characterized mutT locus, which results in A.T----C.G transversions.

A synthetic promoter derived from the Escherichia coli lipoprotein promoter sequence was used to express synthetic suppressor tRNA genes in E. coli. These constructs, cloned into a plasmid of the pEMBL family, gave very high yields of suppressor tRNA in vivo.

A-G mispairs are subject to correction by two distinct pathways in cell-free extracts of Escherichia coli [Su, S.-S., Lahue, R.S., Au, K.G. & Modrich, P. (1988) J. Biol. Chem. 263, 6829-6835; Lu, A.-L. & Chang, D.Y. (1988) Genetics 118, 593-600]. One is the mutHLS-dependent, methyl-directed pathway that recognizes a variety of mismatches and repairs the unmethylated strand of DNA heteroduplexes that are hemimethylated at d(GATC) sequences. The other pathway appears to be specific for A-G mispairs, yields C.G base pairs exclusively, and is independent of the presence of d(GATC) sites. Analyses of cell-free extracts prepared from E. coli mutY strains and isogenic parents have demonstrated that the mutY gene product is involved in the methyl-independent pathway, which converts A-G mispairs to C.G pairs. The specificity of this activity is consistent with the mutator phenotype associated with the mutY locus, which generates G.C----T.A transversions [Nghiem, Y., Cabrera, M., Cupples, C.G. & Miller, J.H. (1988) Proc. Natl. Acad. Sci. USA 85, 2709-2713]. We propose that the mutY product functions at a late stage of a pathway that excludes A-G mispairs during chromosome replication and that involves the function of the mutT gene product. This model suggests that the mutT function acts at an early stage of this pathway to exclude A-G mismatches where the adenine resides on the template DNA strand. A-G mispairs that persist after passage of the replication fork would contain guanine on the template strand and thus be processed to C.G base pairs by the mutY-dependent repair system.

A system for determining the precise specificity of mutagens operating on Escherichia coli has been described, and the results for UV irradiation have been presented. Attempts to correlate the base pairs preferentially mutated by UV light with pyrimidine-pyrimidine sequences are discussed.

We have constructed synthetic genes encoding different Escherichia coli suppressor tRNAs for use in amino acid substitution studies and protein engineering. We used oligonucleotides to assemble the genes for different tRNAs with the anticodon 5' CTA 3'. The suppressor genes are expressed from a synthetic promoter derived from the promoter sequence of the E. coli lipoprotein gene. The genes have been used to suppress an amber mutation in a protein coding sequence, and the resulting altered protein has been subjected to sequence analysis to determine the nature of the amino acid inserted at the amber site. Twelve amino acids can now be added in response to the amber codon. We have employed these suppressors to study amino acid substitutions in the lac repressor.

Three early amber mutations in the lac i gene have been shown to arise from the codons corresponding to residues 7, 12, and 17 of the lac repressor polypeptide chain. All three mutations allow translational reinitiation at the same two sites, resulting in the synthesis of two lac repressor fragments. The amino-terminal sequences of these fragments show that the first site is the triplet coding for valine residue 23, while the second is the first internal in-phase AUG codon corresponding to residue 42. Translational reinitiation appears to be a common event in E. coli, since there are at least three such sites in the first 70 in-phase codons of the i-gene messenger RNA, and all amber mutants found in this region show translational reinitiation. Only one of these sites involves an AUG codon; the other two involve an in vivo ambiguity of the genetic code, in that the same codon can be translated into two different amino acids depending on whether it is recognized during initiation or elongation of protein biosynthesis. The two non-AUG codons are the codons corresponding to leucine residue 62 and valine residue 23 of the lac repressor.

Out of the first 62 residues of the lac repressor, 38 positions have been substituted by at least one amino acid exchange. The total number of replacements in this region is 131. Data from several studies are considered.

Nonsense mutations derived from 90 different codons in the lacI gene of Escherichia coli have been correlated with the I gene nucleotide sequence. In over 80 cases the specific codon which generates the nonsense mutation can be identified. The sequence shows that 14-16 sites arise through tandem double base changes.

We used the lacI system of Escherichia coli to examine the distribution of base substitution mutations occurring spontaneously in different mismatch-repair-deficient strains. The examination of almost 1,200 nonsense mutations generated in strains carrying the mutS, mutH, and mutU alleles confirmed that transitions are highly favored over transversions. The detailed analysis of relative mutation rates at different sites revealed that the pattern of hot spots and cold spots is strikingly similar in each of the three strain backgrounds, strongly supporting the notions that the products of the three genes are part of the same system and that in the absence of any of the components the entire system fails to function. The distribution of mutations occurring in the absence of mismatch repair defined a pronounced topography of the lacI gene. There was no obvious correlation of the hot spots or cold spots with either nearest-neighbor sequences or A X T richness of the immediate surrounding sequence.

We have characterized strains of E. coli in which the lac region, together with varying amounts of surrounding DNA, is amplified 40 to 200 fold. The amplification events involve regions of 7 to 37 kb and result in a tandem array of repeated units. Restriction digest patterns of DNA from over 100 independent strains reveal that the amplified units are different in each case. Mechanisms of gene duplication and amplification, and the relationship of gene amplification in bacteria to that in eucaryotic cells, are considered.