Phage lambda repressor revertants. Amino acid substitutions that restore activity to mutant proteins

We have isolated same-site and second-site revertants that restore partial activity, wild-type activity, or greater than wild-type activity, to lambda repressor proteins bearing different mutations in the DNA binding domain. In some cases the revertant repressors contain same-site substitutions that are similar to the wild-type side-chain (e.g. Tyr22----Phe, Ser77----Thr). The activity of these revertants makes it possible to assess the role of specific hydrogen bonds and/or packing interactions in repressor structure and function. In other same-site revertants, a very different type of residue is introduced (e.g. Ser35----Leu, Gly48----Asn). This indicates that the chemical and steric requirements at these side-chain positions are relaxed. Two of the second-site revertants, Glu34----Lys and Gly48----Ser, restore activity to more than one primary mutant. Both substitutions apparently increase the affinity of the repressor-operator interaction by introducing new contacts with operator DNA. These results suggest that reversion may be a generally applicable method for identifying sequence changes that increase the activity of a protein to greater than wild-type levels.     

The bases of the tRNA anticodon loop are independent by genetic criteria.

We employed two methods to study the translational role of interactions between anticodon loop nucleotides. Starting with a set of previously constructed weakly-suppressing anticodon loop mutants of Su7, we searched for second-site revertants that increase amber suppressor efficiency. Though hundreds of revertants were characterized, no second-site revertants were found in the anticodon loop. Second site reversion was detected in the D-stem, thereby demonstrating the efficacy of the search method. As a second method for detecting interactions, we used site-directed mutagenesis to construct multiple mutations in the anticodon loop. These multiple mutants are very weak suppressors and have translational activities that are equal to or lower than that predicted for the independent action of single mutations. We conclude that although the anticodon loop sequence of Su7 has an optimal structure for the translation of amber codons, we find no evidence that interactions between loop bases can enhance translational efficiency.     

Amino acid substitutions in subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae. Sequence analysis of a series of revertants of an oli1 mit- mutant carrying an amino acid substitution in the hydrophilic loop of subunit 9

This work concerns a biochemical genetic study of subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae. Subunit 9, encoded by the mitochondrial oli1 gene, contains a hydrophilic loop connecting two transmembrane stems. In one particular oli1 mit- mutant 2422, the substitution of a positively charged amino acid in this loop (Arg39----Met) renders the ATPase complex non-functional. A series of 20 revertants, selected for their ability to grow on nonfermentable substrates, has been isolated from mutant 2422. The results of DNA sequence analysis of the oli1 gene in each revertant have led to the recognition of three groups of revertants. Class I revertants have undergone a same-site reversion event: the mutant Met39 is replaced either by arginine (as in wild-type) or lysine. Class II revertants maintain the mutant Met39 residue, but have undergone a second-site reversion event (Asn35----Lys). Two revertants showing an oligomycin-resistant phenotype carry this same second-site reversion in the loop region together with a further amino acid substitution in either of the two membrane-spanning segments of subunit 9 (either Gly23----Ser or Leu53----Phe). Class III revertants contain subunit 9 with the original mutant 2422 sequence, and additionally carry a recessive nuclear suppressor, demonstrated to represent a single gene. The results on the revertants in classes I and II indicate that there is a strict requirement for a positively charged residue in the hydrophilic loop close to the boundary of the lipid bilayer. The precise location of this positive charge is less stringent; in functional ATPase complexes it can be found at either residue 39 or 35. This charged residue is possibly required to interact with some other component of the mitochondrial ATPase complex. These findings, together with hydropathy plots of subunit 9 polypeptides from normal, mutant and revertant strains, led to the conclusion that the hydrophilic loop in normal subunit 9 extends further than previously suggested, with the boundary of the N-terminal membrane-embedded stem lying at residue 34. The possibility is raised that the observed suppression of the 2422 mutant phenotype in class III revertants is manifested through an accommodating change in a nuclear-encoded subunit of the ATPase complex.     

Reversion of an S49 cell cyclic AMP-dependent protein kinase structural gene mutant occurs primarily by functional elimination of mutant gene expression.

The regulatory subunits of cyclic AMP (cAMP)-dependent protein kinase from a dibutyryl cAMP-resistant S49 mouse lymphoma cell mutant, clone U200/65.1, and its revertants were visualized by two-dimensional polyacrylamide gel electrophoresis. Clone U200/65.1 co-expressed electrophoretically distinguishable mutant and wild-type subunits (Steinberg et al., Cell 10:381-391, 1977). In all 48 clones examined, reversion of the mutant to dibutyryl cAMP sensitivity was accompanied by alterations in regulatory subunit labeling patterns. Some spontaneous (3 of 11) and N-methyl-N'-nitro-N-nitrosoguanidine-induced (2 of 11) revertants retained mutant subunits, but these were altered in charge, degree of phosphorylation, or both. The charge alterations were consistent with single amino acid substitutions, suggesting that reversion was the result of second-site mutations in the mutant regulatory subunit allele that restored wild-type function, although not wild-type structure, to the gene product. The majority of spontaneous (8 of 11) and N-methyl-N'-nitro-N-nitrosoguanidine-induced (9 of 11) revertants and all of the revertants induced by ethyl methane sulfonate (14 of 14) and ICR191 (12 of 12) displayed only wild-type subunits. Dibutyryl cAMP-resistant mutants isolated from several of these revertants displayed new mutant but not wild-type subunits, suggesting that the revertant parent expresses only a single, functional regulatory subunit allele. The mutant regulatory subunit allele can, therefore, be modified in two general ways to produce revertant phenotypes: (i) by mutations that restore its wild-type function, and (ii) by mutations that eliminate its function.     

The Neurospora am gene and NADP-specific glutamate dehydrogenase: mutational sequence changes and functional effects--more mutants and a summary

A further series of mutant am alleles, encoding potentially active NADP-specific glutamate dehydrogenase (GDH) and capable of complementation in heterocaryons, have been characterized with respect to both GDH properties and DNA sequence changes. Several mutants previously studied, and some of their same-site or second-site revertants, have also been sequenced for the first time. We present a summary of what is known of the properties of all am mutants that have been defined at the sequence level.     

Isolation and characterization of Escherichia coli birA intragenic suppressors

Summary The biotin (bio) operon in Escherichia coli is negatively regulated by BirA, a bifunctional protein with both repressor and biotin-activating functions. Twenty-five heatresistant revertants of three temperature-sensitive birA alleles (birA 85, bir A 104 and bir A 879) were isolated and categorized into five growth and six repression classes. The revertants appear to increase biotin activation by raising the specific activity of BirA and/or, increasing the number of enzyme molecules. The 19 bir A 85 revertants displayed a broad range of activity for both enzyme and repressor functions, and may represent intragenic second-site suppressor mutations. The bir A 85 revertants included a novel class of bio superrepressor mutations. Repressor titration experiments suggested that many of the bir A 85 revertants increase BirA concentrations above wild-type levels because the repressors were not competed from the chromosomal bio operator by multicopy bio operator plasmids. The majority of the bir A 104 revertants resulted in both wild-type repressor and enzyme activity; they are possibly true revertants in which the amino acid residue altered by the bir A 104 mutation has been substituted by the wild-type or a chemically similar amino acid.     

Lambda repressor mutations that increase the affinity and specificity of operator binding

Intragenic, second-site reversion has been used to identify amino acid substitutions that increase the affinity and specificity of the binding of lambda repressor to its operator sites. Purified repressors bearing the second-site substitutions bind operator DNA from 3 to 600 fold more strongly than wild type; these affinity changes result from both increased rates of operator association and decreased rates of operator dissociation. Three of the revertant substitutions occur in the alpha 2 and alpha 3 DNA binding helices of repressor and seem to increase affinity by introducing new salt-bridges or hydrogen bonds with the sugar-phosphate backbone of the operator site. The fourth substitution alters the alpha 5 dimerization helix of repressor and appears to increase operator affinity indirectly.     

Specific induction of transitions and transversions of G-C base pairs by 4-nitroquinoline-1-oxide in iso-1-cytochrome c mutants of yeast

The base-pair changes induced by the highly carcinogenic agent, 4-nitroquinoline-1-oxide, have been determined from the reversion rates of defined tester strains and from the amino acid replacements of revertant iso-1-cytochromes c. The mutant codons and the base-pair changes of reverse mutations of 14 cyc1 mutants were previously determined from alterations of iso-1-cytochromes c in intragenic revertants. These 14 cyc1 mutants, which were used as tester strains, included nine mutants with altered AUG initiation codons, an ochre (UAA) mutant, an amber (UAG) mutant and three frameshift mutants (Stewart et al., 1971,1972; Stewart &; Sherman, 1972,1974; Sherman &; Stewart, 1973). NQO² induced a high rate of reversion in the initiation mutant cyc1-131, the only mutant in the group which reverts to normal iso-1-cytochrome c by a G · C → A · T transition. In addition, NQO produces a significant rate of reversion of all cyc1 mutants which revert by G · C transversions, e.g. the amber (UAG) mutant and the initiation mutants containing AGG, and probably CUG mutant codons. It did not revert the ochre mutant which contains no G · C base pairs. Ten NQO-induced revertants of the amber mutant cyc1-179 contained the expected replacements of residues of tyrosine, and ten NQO-induced revertants of each of the cyc1-131 and cyc1-133 initiation mutants all contained the expected normal iso-1-cytochrome c. The structures of these iso-1-cytochromes c and the pattern of reversion of the tester strains indicate that base-pair substitutions arise at G · C base pairs which are the site of NQO attack. Thus NQO induces G · C → A · T transitions, G · C → T · A transversions and possibly G · C → C · G transversions. Because of its mode of action, NQO may be useful in less-defined systems for identifying G · C base pairs in mutant codons.     

Transformation-defective virus mutants in a class of morphologic revertants of sarcoma virus transformed nonproducer cells

In studies of the viral and cellular functions involved in expression of transformation by murine sarcoma virus, selective methods have led to the isolation of morphologic revertants following mitomycin C mutagenization of nonproductively transformed mouse cells. The revertants exhibit normal growth properties, yet still contain the sarcoma virus. Further, they are as susceptible as normal cells to exogenous sarcoma virus infection. In the present studies, these revertants are shown to contain levels of sarcoma viral RNA quantitatively and qualitatively indistinguishable from that present in the parental transformed clone. Following rescue with helper leukemia virus, they release low levels of wild-type transforming virus and a large excess of transformation-defective sarcoma virus as measured by molecular hybridization. The defective viruses can be transmitted to new cells in the absence of morphologic alteration. These results provide strong evidence that the revertants contain mutant viruses defective in transforming functions. The release of wild-type sarcoma virus by cells in a revertant culture appears to occur concomitantly with the spontaneous appearance of retransformed cells. This suggests that the reversion of mutant virus to wild-type within the cell occurs as a result of reversion of a point mutation in the integrated sarcoma viral genome. The present sarcoma virus mutants appear to be the first obtained by spontaneous or chemically-induced genetic alteration of stably integrated virus in eucaryotic cells.     

Isolation and characterization of revertants from four different classes of aryl hydrocarbon hydroxylase-deficient hepa-1 mutants.

Revertants were selected from aryl hydrocarbon hydroxylase (AHH)-deficient recessive mutants belonging to three complementation groups and from a dominant mutant of the Hepa-1 cell line. The recessive mutants had low spontaneous reversion frequencies (less than 4 X 10(-7] that were increased by mutagenesis. The majority of these revertants also had reacquired only partial AHH activity. Revertants of group A mutants were identical to the wild type with respect to both in vivo and in vitro enzyme stability and the Km for the substrate, benzo [alpha]pyrene, and therefore failed to provide evidence that gene A is the AHH structural gene. Group B and group C mutants are defective in the functioning of the Ah receptor required for AHH induction. Revertants of these groups were normal with respect to in vivo temperature sensitivity for AHH induction and for the 50% effective dose for the inducer, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and thus provided no evidence that the B and C genes code for components of the receptor. Two rare group C revertants possessed AHH activity in the absence of induction. The phenotype of one of these was shown to be recessive to the wild type. Spontaneous revertants of the dominant mutant occurred at a frequency 300-fold greater than those of the recessive mutants, and this frequency was not increased by mutagenesis. These revertants all displayed complete restoration of AHH activity to wild type levels. These observations and the results from cell hybridization studies suggest that the dominant revertants arose by a high frequency event leading to functional elimination of the dominant mutation.     

Distribution of mannosamine and mannosaminuronic acid among cell walls of Bacillus species

Some Escherichia coli K-12 lamB mutants, those producing reduced amounts of LamB protein (one-tenth the wild type amount), grow normally on dextrins but transport maltose when present at a concentration of 1 microM at about one-tenth the normal rate. lamB Dex- mutants were found as derivatives of these strains. These Dex- mutants are considerably impaired in the transport of maltose at low concentrations (below 10 microM), and they have a structurally altered LamB protein which is impaired in its interaction with phages lambda and K10 but still interacts with a lambda host range mutant lambda hh*. The Dex- mutants are double lamB mutants carrying one mutation, already present in the parental strains, that reduces LamB synthesis and a second that alters LamB structure. The secondary mutations, present in different independent Dex- mutants, are clustered in the same region of the lamB gene. Dex+ revertants were isolated and analyzed: when the altered LamB protein is made in wild-type amount, due to a reversion of the first mutation, the phenotype reverts to Dex+. However, these Dex+ revertants are still very significantly impaired in maltose transport at low concentrations (below 10 microM).     

Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.     

Genetic analysis of mutants of Aspergillus nidulans blocked at an early stage of sporulation.

Three mutants of Aspergillus nidulans, selected to have a block at an early stage of conidiation (asexual sporulation), exhibit similar pleiotropic phenotypes. Each of these mutants, termed preinduction mutants, also are blocked in sexual sporulation and secrete a set of phenolic metabolites at level much higher than wild type or mutants blocked at later stages of conidiation. Backcrosses of these mutants to wild type showed that the three phenotypes always cosegregated. Diploids containing the mutant alleles in all pairwise combinations were normal for all phenotypes, showing that the three mutations are nonallelic. This conclusion was confirmed by the finding that the mutations map at three unlinked or distantly linked loci. Ten revertants of the two least leaky preinduction mutants, selected for ability to conidiate, were found in each case to arise by a second-site suppressor mutation. All of the revertants still showed accumulation of some of the phenolic metabolites but differed from each other in certain components. Three of the revertants retained the block in sexual sporulation. In these cases the suppressor has thus uncoupled the block in asexual sporulation from the block in sexual sporulation. These results are understandable in terms of a model in which preinduction mutations and their suppressors affect steps in a single metabolic pathway whose intermediates include an effector specific for asexual sporulation and a second effector specific for sexual sporulation.     

Genetic Analysis of Bacteriophage P22 Lysozyme Structure

The suppression patterns of 11 phage P22 mutants bearing different amber mutations in the gene encoding lysozyme (19) were determined on six different amber suppressor strains. Of the 60 resulting single amino acid substitutions, 18 resulted in defects in lysozyme activity at 30 degrees; an additional seven were defective at 40 degrees. Revertants were isolated on the "missuppressing" hosts following UV mutagenesis; they were screened to distinguish primary- from second-site revertants. It was found that second-site revertants were recovered with greater efficiency if the UV-irradiated phage stocks were passaged through an intermediate host in liquid culture rather than plated directly on the nonpermissive host. Eleven second-site revertants (isolated as suppressors of five deleterious substitutions) were sequenced: four were intragenic, five extragenic; three of the extragenic revertants were found to have alterations near and upstream from gene 19, in gene 13. Lysozyme genes from the intragenic revertant phages were introduced into unmutagenized P22, and found to confer the revertant plating phenotype.     

The interaction between aspartic acid 237 and lysine 358 in the lactose carrier of Escherichia coli.

The lacY from Escherichia coli strains 020 and AE43 have been cloned on plasmids which were designated p020-K358T and pAE43-D237N. These lacY mutants contain amino acid substitutions changing Lys-358 to Thr or Asp-237 to Asn, respectively. The charge neutralizing effect of each mutation is associated with a functional defect in melibiose transport which we exploited in order to isolate second site revertants to the melibiose-positive phenotype. Eleven melibiose-positive revertants of p020-K358T were isolated. All contained a second-site mutation converting Asp-237 to a neutral amino acid (8 to Asn, 1 to Gly, and 2 to Tyr). Twelve melibiose-positive revertants of pAE43-D237N were isolated. Two were second-site revertants converting Lys-358 to a neutrally Gln residue, while the remainder directly reverted Asn-237 to the wild-type Asp-237. We conclude that the functional intimate relationship between Asp-237 and Lys-358 suggests that these residues may be closely juxtaposed in three-dimensional space, possibly forming a 'charge-neutralizing' salt bridge.     

A Search for a General Phenomenon of Adaptive Mutability

The most prominent systems for the study of adaptive mutability depend on the specialized activities of genetic elements like bacteriophage Mu and the F plasmid. Searching for general adaptive mutability, we have investigated the behavior of Salmonella typhimurium strains with chromosomal lacZ mutations. We have studied 30 revertible nonsense, missense, frameshift, and insertion alleles. One-third of the mutants produced >=10 late revertant colonies (appearing three to seven days after plating on selective medium). For the prolific mutants, the number of late revertants showed rank correlation with the residual β-galactosidase activity; for the same mutants, revertant number showed no correlation with the nonselective reversion rate (from fluctuation tests). Leaky mutants, which grew slowly on selective medium, produced late revertants whereas tight nongrowing mutants generally did not produce late revertants. However, the number of late revertants was not proportional to residual growth. Using total residual growth and the nonselective reversion rate, the expected number of late revertants was calculated. For several leaky mutants, the observed revertant number exceeded the expected number. We suggest that excess late revertants from these mutants arise from general adaptive mutability available to any chromosomal gene.     

Deletion and amplification of the HGPRT locus in Chinese hamster cells.

Somatic cell selective techniques and hybridization analyses with a cloned cDNA probe were used to isolate and identify Chinese hamster cell lines in which the X-linked gene for hypoxanthine-guanine phosphoribosyltransferase (HGPRT) has been altered. Two of 19 HGPRT-deficient mutants selected were found to have major DNA deletions affecting the HGPRT locus. Cytogenetic studies revealed that the X chromosome of each deletion mutant had undergone a translocation event, whereas those from the remaining 17 mutants were normal. Phenotypic revertants of the thermosensitive HGPRT mutant RJK526 were isolated, and amplification of the mutant allele was shown to be the predominant mechanism of reversion. Comparisons of restriction enzyme fragments of DNA from deletion versus amplification strains identified two regions of the Chinese hamster genome that contained homology to the cDNA probe. One was shown to be much larger than the 1,600-nucleotide mRNA for HGPRT and to be comprised of linked fragments that contained the functional HGPRT gene. The second was neither transcribed nor tightly linked to the functional gene. These initial studies of HGPRT alterations at the level of DNA thus identified molecular mechanisms of phenotypic variation.     

Paclitaxel resistance in cells with reduced beta-tubulin

We previously described the isolation of colcemid resistant Chinese hamster ovary cell lines containing alpha- and beta-tubulin mutations that increase microtubule assembly and stability. By analyzing colcemid sensitive revertants from one of the beta-tubulin mutants, we now find that loss or inactivation of the mutant allele represents the most common mechanism of reversion. Consistent with this loss, the revertants have 35% less tubulin at steady state, no evidence for the presence of a mutant polypeptide, and a normal extent of tubulin polymerization. In addition to the loss of colcemid resistance, the revertant cells exhibit increased resistance to paclitaxel relative to wild-type cells. This paclitaxel resistance can be suppressed by transfecting the revertant cells with a cDNA for wild-type beta-tubulin, indicating that the reduction in tubulin in the revertant cells is responsible for the resistance phenotype. We propose that reducing tubulin levels may represent a novel mechanism of paclitaxel resistance.     

Simian virus 40 mutant T antigens with relaxed specificity for the nucleotide sequence at the viral DNA origin of replication.

Base substitution of the ori region of simian virus 40 leads to plaque morphology mutants with markedly decreased DNA replication. Second-site mutations within the simian virus 40 T antigen gene suppress the plaque phenotype and replication defect of base-substituted ori mutants. Two second-site mutations have been mapped to a small segment of the T antigen gene, just beyond the distal splice junction. DNA sequence analysis revealed a single missense change in this segment of the T antigen gene of each of these second-site revertants, leading to a change in codon 157 in one case and codon 166 in the other. The mutant T antigens displayed relaxed specificity for the ori signal, i.e., they can function with several variously modified ori sequences, including those with small nucleotide deletions or insertions that are inactive for replication when coupled with wild-type T antigen. Thus a region of T antigen has been identified that appears to be intimately involved in vivo in binding to the ori sequence to initiate viral DNA replication.     

Suppressors of Cleavage-Site Mutations in the p62 Envelope Protein of Semliki Forest Virus Reveal Dynamics in Spike Structure and Function

The E2 spike glycoprotein of Semliki Forest virus is produced as a p62 precursor protein, which is cleaved by host proteases to its mature form, E2. Cleavage is not necessary for particle formation or release but is necessary for infectivity. Previous results had shown that phenotypic revertants of cleavage-deficient p62 mutants are generated, and here we show that these may contain second-site suppressor mutations in the vicinity of the cleavage site. These hot-spot sites were mutated to abolish the generation of such suppressor mutations; however, secondary mutations in another distant domain of the E2 protein appeared instead, all of which still caused cleavage-deficient mutations. Such mutants grew very poorly and were inefficient in virus entry and release. The mutated sites define domains of the spike protein which probably interact to regulate its structure and function. Because of their highly attenuated phenotype and the lower probability of reversion, the new mutations close to the cleavage site were used to make new helper vectors for packaging of recombinant RNA into infectious particles, thus increasing further the biosafety of the vector system based on the Semliki Forest virus replicon.