Frequency of spontaneous mutations in an avian hepadnavirus infection

In this study, we measured the frequency of revertants of a cytopathic strain of the duck hepatitis B virus that bears a single nucleotide substitution in the pre-S envelope protein open reading frame, resulting in the amino acid substitution G133E. Cytopathic virus mixed with known amounts of a genetically marked wild-type virus was injected into ducklings. Virus outgrowth was accompanied by a coselection of wild-type and spontaneous revertants during recovery of the ducklings from the acute liver injury caused by death of the G133E-infected cells. The frequency of individual revertants in the selected noncytopathic virus population was estimated by determining the ratio of each revertant to the wild-type virus. Spontaneous revertants were found to be present at frequencies of 1 x 10(-5) to 6 x 10(-5) per G133E genome inoculated. A mathematical model was used to estimate that the mutation rate was 0.8 x 10(-5) to 4.5 x 10(-5) per nucleotide per generation.     

Revertants of v-fos-transformed fibroblasts have mutations in cellular genes essential for transformation by other oncogenes

Morphologic revertants of FBJ murine sarcoma virus (v-fos)-transformed rat-1 fibroblasts were isolated using a novel selection procedure based on prolonged retention of rhodamine 123 within mitochondria of v-fos-transformed versus normal fibroblasts. Two classes of revertants were isolated: class I revertants have sustained mutations in cellular genes, and a class II revertant has a nonfunctional v-fos provirus. Somatic-cell hybridization studies suggested that the revertant phenotype was recessive to the transformed phenotype. Class I revertants were also resistant to retransformation by v-gag-fos-fox, v-Ha-ras, v-abl, and v-mos, but could be retransformed by the trk oncogene and polyoma virus middle T antigen. These results suggest that the class I revertants sustained mutations in one or more cellular genes essential for transformation by some, but not all, oncogenes. Our data suggest the existence of common biochemical pathways for transformation.     

Genetic events associated with an insertion mutation in yeast

The his4-912 mutation shares similar genetic properties with mutations promoted by procaryotic insertion elements. This mutation lacks all three his4 functions. Many different classes of His+ revertants have been obtained from his4-912. The most frequent class of His+ revertants results from a site mutation which confers a cold-sensitive His- phenotype. Other classes of revertants contain translocations (one between chromosomes I and III and the other between chromosomes III and XII), a transposition of the his4 region to chromosome VIII, and an inversion of most of the left arm of chromosome III. Another class contains deletions which extend from his4-912 into the his4 region. In each of these classes of revertants, the his4 region is closely linked to the chromosomal aberration. Many of these revertants contain additional changes in chromosome structure (duplication, deletion and aneuploidy) that are unrelated to the reversion of his4-912 to His4+.     

Genomic rearrangements in the flagellin genes of Proteus mirabilis

Molecular analyses have revealed that Proteus mirabilis possesses two genes, flaA and flaB, that are homologous to each other and to flagellin genes of many other species. Both swimmer and swarmer cells transcribe flaA, but not flaB. FlaA- mutants are non-motile and do not differentiate showing the essential role of flaA in swarmer cell differentiation and behaviour. At a low frequency, motile, differentiation-proficient revertants have been found in FlaA-populations. These revertants produce an antigenically and biochemically distinct flagellin protein. The revertant flagellin is the result of a genetic fusion between highly homologous regions of flaA and flaB that places the active flaA promoter and the 5' coding region of flaA adjacent to previously silent regions of flaB generating a hybrid flagellin protein. Analysis of the flaA-flaB region of two such revertants reveals that a portion of this locus has undergone a rearrangement and deletion event that is unique to each revertant. Using a polymerase chain reaction (PCR) to amplify the falA-flaB locus from wild-type swimmer cells, swarmer cells and cells obtained after urinary tract infection, we uncover at least six general classes of rearrangements between flaA and flaB. Each class of rearrangement occurs within one of nine domains of homology between flaA and flaB. Rearrangement of flaA and flaB results in a hybrid flagellin protein of nearly identical size and biochemical properties, suggesting a concerted mechanism may be involved in this process. The data also reveal that the frequency and distribution of flaAB rearrangements is predicted on environmental conditions. Thus, rearrangement between flaA and flaB may be a significant virulence component of P. mirabilis in urinary tract infections.     

Role of the 5' hairpin structure in the splicing accuracy of the fourth intron of the yeast cob-box gene

The splicing mechanism of the maturase-coding introns is poorly understood. We have systematically examined the phenotypes of a large number of revertants from the mitochondrial mutation G2457. This mutation results from a single base change near the 5' splicing site. We show here that this base change does not completely block the splicing of the intron but rather affects the specificity of the splicing process. We examine four classes of revertants which allow us to characterize the crucial role of a stem and loop structure in the accuracy of the intron excision process. An unexpected class of revertant suggests that other elements are involved in this mechanism. Reversion of G2457 can also occur via the excision in the mitochondrial genome of the intron coding sequence. These results are discussed in relation to the possible role fulfilled by the maturase in the control of intron splicing.     

ABI1 protein phosphatase 2C is a negative regulator of abscisic acid signaling.

The plant hormone abscisic acid (ABA) is a key regulator of seed maturation and germination and mediates adaptive responses to environmental stress. In Arabidopsis, the ABI1 gene encodes a member of the 2C class of protein serine/threonine phosphatases (PP2C), and the abi1-1 mutation markedly reduces ABA responsiveness in both seeds and vegetative tissues. However, this mutation is dominant and has been the only mutant allele available for the ABI1 gene. Hence, it remained unclear whether ABI1 contributes to ABA signaling, and in case ABI1 does regulate ABA responsiveness, whether it is a positive or negative regulator of ABA action. In this study, we isolated seven novel alleles of the ABI1 gene as intragenic revertants of the abi1-1 mutant. In contrast to the ABA-resistant abi1-1 mutant, these revertants were more sensitive than the wild type to the inhibition of seed germination and seedling root growth by applied ABA. They also displayed increases in seed dormancy and drought adaptive responses that are indicative of a higher responsiveness to endogenous ABA. The revertant alleles were recessive to the wild-type ABI1 allele in enhancing ABA sensitivity, indicating that this ABA-supersensitive phenotype results from a loss of function in ABI1. The seven suppressor mutations are missense mutations in conserved regions of the PP2C domain of ABI1, and each of the corresponding revertant alleles encodes an ABI1 protein that lacked any detectable PP2C activity in an in vitro enzymatic assay. These results indicate that a loss of ABI1 PP2C activity leads to an enhanced responsiveness to ABA. Thus, the wild-type ABI1 phosphatase is a negative regulator of ABA responses.     

M13 DNA fingerprinting can be used in studies on phenotypic reversions of forest tree mutants

 Solitary revertants which have been observed on single mutant tree individuals have up to now been believed to be grow-through cells belonging to the rootstock on which they are commonly grafted. In this study three different phenotypically visible mutants revealing revertant shoots on the same tree were chosen for genetic analysis. The mutant Quercus robur L. ‘argenteomarginata’ was grafted on a normal rootstock, an individual of Carpinus betulus L. var. quercifolia Desf. as well as an individual of Picea glauca (Moench) Voss. ‘conica’ are supposed to have grown from seeds. By means of a highly specific M13 PCR fingerprinting technique the mutant and revertant tissues were analysed in comparison to different individuals of each of the species. With the grafted mutant, cambium tissue of the rootstock was also investigated. Whereas conspecific individuals could be clearly distinguished from each other, mutant and revertant tissues revealed the same banding patterns for each of the three trees. In case of the grafted mutant, the fingerprint obtained from cambium tissue of the rootstock was clearly different from the pattern of mutant and revertant tissue. Results demonstrate the potential of the tool for genetic differentiation between individuals of three tree species hence in the case of the grafted mutant, the hypothesis that the observed reversion is caused by a grow-through of the rootstock is rejected. Furthermore, identical fingerprints of mutant and revertant tissue support identical genetic background of the tissues excluding the gene(s) responsible of the mutation. Possible causes of mutations and reversions regarding the three mutant trees are discussed. Received: 15 September 1997 / Accepted: 24 November 1997     

Reversion at the HIS1 Locus of Yeast

The his1 gene (chromosome V) of Saccharomyces cerevisiae specifies phosphoribosyl transferase (E.C.2.4.2.17), the first enzyme of histidine biosynthesis. This hexameric enzyme has both catalytic and regulatory functions. The spontaneous reversion rates of seven his1 mutations were studied. The reversion rates of the alleles at the proximal end of the locus (relative to the centromere) were about 50-fold higher than distal alleles. Spontaneous reversion to prototrophy was studied in diploids homoallelic for each of the seven his1 mutations. Based on tetrad analysis, the prototrophy revertants could be assigned to three classes: (1) revertant tetrads that carried a prototrophic allele indistinguishable from wild type; (2) revertant tetrads that carried a prototrophic allele characterized by histidine excretion and feedback resistance; and (3) revertant tetrads that did not contain a prototrophic spore, but rather a newly derived allele that complemented the original allele intragenically. Four of the seven his1 mutations produced the excretor revertant class, and two mutations produced the complementer revertant class. The significance of these findings to our understanding of gene organization and the catalytic and regulatory functions of gene products are discussed.     

Characterization of Morphologic Revertants of Murine and Avian Sarcoma Virus-Transformed Cells

Morphologic revertants which contain avian or murine sarcoma viruses have previously been isolated at low frequency from clonal lines of transformed mammalian cells. In the present study, these lines have been further characterized. They are indistinguishable from nontransformed parent cell lines with respect to parameters such as saturation density and colony formation in depleted medium or on monolayers of contact-inhibited cells. The rate of glucose uptake had also reverted to normal. The malignant potential of one of the revertant lines was examined and found to be markedly reduced compared to that of the corresponding transformed cells. The differences in the susceptibilities of revertant cells to retransformation by the same or other oncogenic viruses suggest that different cellular genes may be involved in expression of transformation by various tumor viruses.     

Plasmid-dosage effects on ultraviolet, visible light and methyl methanesulfonate mutagenesis in Salmonella typhimurium

Salmonella typhimurium LT2 strains bearing plasmids pKM101, R64 or pColIb-P9 demonstrated enhanced UV survival when compared with strains not bearing plasmids. A strain of S. typhimurium bearing both pKM101 and pColIb-P9 survived UV irradiation slightly better than either of the single-plasmid strains. Spontaneous reversion of the hisG46 and trpE8 missense alleles was enhanced in each single-plasmid strain, and for the dual-plasmid strain containing pKM101 and pColIb-P9 enhancement represented a near additivity of the response seen for the single-plasmid strains. Following exposure to UV or visible-light irradiation, reversion of hisG46 and trpE8 was also enhanced in each single-plasmid strain, but quantitatively greater in the dual-plasmid strain and was equal to or slightly greater than additive the responses of the single-plasmid strains. In contrast to visible-light irradiation, UV exposure resulted in two phenotypic Trp+-revertant classes. One Trp+ class, having normal colony size (2.0 mm) and similar in number to His+ revertants, was comprised of intragenic revertants of trpE8, while the predominant Trp+ class, having smaller colony size (0.8 mm), represented intergenic suppressor revertants, illuminating the differences in mutation and/or repair specificity for UV and visible-light exposure. Methyl methane-sulfonate (MMS)-induced reversion of hisG46 was similar in effect to that seen with UV or visible-light irradiation. Plasmids pKM101 or pColIb-P9 enhanced the frequency of hisG46 reversion, while a more than additive response was seen in a strain with both plasmids. Furthermore, MMS-induced reversion of hisG46 was also observed to be greatest in a strain bearing plasmid R64 (incompatibility group I alpha) and pKM101, when compared with single-plasmid strains bearing either R64 or pKM101.     

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.     

An Unstable Allele of the am Locus of NEUROSPORA CRASSA

The mutant strain am126 was isolated, using the direct selection procedure, after nitrous acid mutagenesis. It produced neither measurable NADP-dependent glutamate dehydrogenase (GDH) nor immunologically cross-reacting material. That the am126 strain produced some form of GDH product was shown by the fact that it complemented several other am mutant strains. The GDH formed by complementation between am126 and each of two other am mutants was relatively thermolabile, but could not be distinguished from wild-type GDH formed by electrophoresis in polyacrylamide gels. This, together with the relatively high yield of the complementation enzymes, suggest that the am126 product is a polypeptide chain not grossly abnormal in structure. The spontaneous revertant frequency was between 0.3 and 3 prototrophic revertants per 10(5) live cells. This frequency was at least 40 times greater than that for am19, which had the second highest spontaneous revertant frequency among the mutants tested. Neither meiosis nor mutagenesis increased the revertant frequency, nor did incubation at elevated temperatures lower it. Sixty-eight revertant strains were examined for thermostability of their GHD. All appeared to be identical to wild type. Seven of the revertant strains were also tested for instability with regard to forward mutation to am auxtrophy. None was found to be unstable. Models for the genetic instability of the am126 mutation are discussed.     

Interaction of super-repressible and dominant constitutive mutations for the synthesis of galactose pathway enzymes in Saccharomyces cerevisiae.

Two dominant uninducible mutant alleles in the gal80 locus were identified. The GAL80s-1 and GAL80s-2 mutants showed novel phenotypes in response to the newly isolated GAL81-1 mutant allele, a dominant constitutive mutation linked to the gal4 locus; the GAL80s-1 GAL81-1 strain was inducible and the GAL80s-2 GAL81-1 strain was uninducible. Many galactose positive revertants from the GAL80s-2 GAL81-1 strain were isolated. It was proved that each revertant was due to a secondary mutation either in the gal80 or GAL81 locus, whereas revertants due to mutation at the supposed controlling site for the structural gene cluster of the galactose-pathway enzymes have not been isolated.     

Properties of mutants of Escherichia coli lacking malic dehydrogenase and their revertants.

Mutants of Escherichia coli lacking malic dehydrogenase activity (mdh) were incapable of growth on acetate", succinate- or malate/mineral medium. Revertants of mdh strains which had regained the ability to grow on C4-dicarboxylic acids could be divided into two distinct classes. One type of revertant had regained the ability to synthesize functional malic dehydrogenase. The other type of revertant still lacked malic dehydrogenase activity but possessed a suppressor mutation which altered the regulation of the synthesis or activity of the C4-dicarboxylic acid transport system, resulting in increased C4-dicarboxylic acid transport activity. This latter class of revertants apparently synthesized oxalacetate from malate via the sequential actions of the NAD-linked malic enzyme, phosphoenolpyruvate synthetase, and phosphoenolpyruvate carboxylase. Evidence has been presented that is consistent with the hypothesis that oxalacetate is the inducer of the C4-dicarboxylic acid transport system. The inability of mutants lacking malic dehydrogenase to grow with a C4-dicarboxylic acid as the carbon source can be attributed to the difficulty such mutants have in synthesizing oxalacetate.     

Search for revertants of the glutamine mischarging mutans of Escherichia coli su+3 tyrosine suppressor tRNA that are able to insert tyrosine at the site of amber mutation.

We have isolated a bacterial amber mutation (nadam) that is suppressed by the tyrosine inserting suppressor su+3 but not by the glutamine (su+2, su+3 A1, su+3 G82 and su+3 A1G82), serine (su+1) and leucine (su+6) inserting suppressors. The su+7 suppressor which inserts glutamine and tryptophan also suppresses this mutation indicating that tryptophan, in addition to tyrosine, is accepted at the site of amber mutation. We have used this amber mutation to search for revertants of the su+3 glutamine mischarging mutants su+3 A1, su+3 G82 and su+3 A1G82 that are able to insert tyrosine at the site of amber mutation. Two types of revertants were found in the case of su+3 A1. One type corresponding to the true revertant A1 leads to G, and the other to the second site revertants C81 leads to U (A1U81). The A1U81 revertant has been shown to insert both glutamine and tyrosine at the site of amber mutation. Only true revertants (G82 leads to A) were obtained when su+3 G82 was analyzed. No revertants were obtained in the case of the su+3 A1G82. These results are discussed in relation to aminoacyl-tRNA recognition.     

Evidence for coupling between membrane and cytoplasmic domains of the yeast plasma membrane H(+)-ATPase. An analysis of intragenic revertants of pma1-105.

A genetic approach was used to identify interacting portions of the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae. The cellular sensitivity of the pma1-105 strain (S368F) to low external pH and to NH4+ was used to select intragenic revertants of two classes: phenotypically wild-type full revertants and partial revertants that were low pH-resistant but retained resistance to hygromycin B. All 10 full revertants had S368 restored. Among five partial revertants mapping to the original site within the phosphorylation domain, S368L and S368V were each found twice. One revertant contained an E367V substitution adjacent to the original S368F alteration. Four of 13 independently isolated second-site revertants mapped to one site, V289F, in the proposed phosphatase domain. Mutations within the proposed phosphatase and phosphorylation domains resulted in enzymes with increased vanadate sensitivity relative to the vanadate-insensitive S368F enzyme. These results suggest that sites S368, E367, and V289 contribute to a vanadate (Pi) binding domain or are able to interact with such a site within the catalytic domain. The remaining nine partial second-site revertants mapped to six sites within the putative transmembrane regions. Mutations within the transmembrane region had less of an effect on vanadate sensitivity. Most revertant enzymes showed small but significant increases in the rate of ATP hydrolysis relative to the S368F enzyme. Several enzymes no longer displayed the acid-sensitive pH-dependence seen in the S368F enzyme. These data provide novel evidence for an interaction between putative transmembrane helices 1-3 and 7 and the ATP hydrolytic portion of the enzyme.     

Association of mutator activity with UV sensitivity in an aphidicolin-resistant mutant of Chinese hamster V79 cells

The spontaneous mutation rates of an ultraviolet light (UV)-sensitive aphidicolin-resistant mutant (aph^r-4-2) and its revertants have been determined by 2 techniques. By using the fluctuation analysis, the mutant and its thymidine (TdR)-prototrophic ‘revertant’ were found to exhibit elevated spontaneous mutation rates at the 6-thioguanine- and diphtheria-toxin-resistant loci. In constrast, the TdR-auxotrophic ‘revertant’ did not show this property. Similar results were obtained by the multiple replating technique. From these comparative studies and other previous characterizations, it appears that a single gene mutation is responsible for the following pleiotropic phenotype: slow growth, UV sensitivity, high UV-induced mutability, high frequency of site-specific bromodeoxyuridine (BrdU)-dependent chromosome breaks and enhanced spontaneous mutation rate. Recent studies indicate that the mutation may be on the gene for DNA polymerase α. The results further indicate that thymidine auxotrophy or imbalance in nucleotide poolsis not necessarily associated with the mutator activity in mammalian cells.     

Evaluation of the genetic stability of the temperature-sensitive PB2 gene mutation of the influenza A/Ann Arbor/6/60 cold-adapted vaccine virus.

A single-gene reassortant bearing the PB2 gene of the A/Ann Arbor/6/60 cold-adapted virus in the background of the A/Korea/82 (H3N2) wild-type virus is a temperature-sensitive (ts) virus with an in vitro shutoff temperature of 38 degrees C. A single mutation at amino acid (aa) at 265 (Asp-Ser) of the PB2 protein is responsible for the ts phenotype. This ts single-gene PB2 reassortant virus was serially passaged at elevated temperatures in Madin-Darby canine kidney cells to generate ts+ phenotypic revertant viruses. Four ts+ phenotypically revertant viruses were derived independently, and each possessed a shutoff temperature for replication in vitro of > 40 degrees C. Each of the four phenotypically revertant viruses replicated efficiently in the upper and lower respiratory tracts of mice and hamsters, unlike the PB2 single-gene reassortant virus, confirming that the ts phenotype was responsible for the attenuation of this virus in rodents. Mating the ts+ revertants with wild-type virus yielded ts progeny in high frequency, indicating that the loss of ts phenotype was due to a suppressor mutation which was mapped to the PA gene in each of the four independently derived ts phenotypic revertants. Nucleotide sequence analysis confirmed the absence of new mutations on the PB2 gene and the presence of predicted amino acid changes in the PA proteins of the revertant viruses. These studies suggest that single amino acid changes at aa 245 (Glu-Lys) or 347 (Asp-Asn) of the PA protein can completely suppress the ts and attenuation phenotypes specified by the Asp-Ser mutation at aa 265 of the PB2 protein of the A/Ann Arbor/6/60 cold-adapted virus.     

Pectinolytic activity of revertants of auxotrophic strains of Aspergillus niger

From conidia of 4 different auxotrophic A. niger strains 400 spontaneous revertants (100 from each strain) were obtained, and in one case additionally 100 revertants induced by mutagens (UV+NTG). The revertants showed a considerable differentiation with regard to the total pectinolytic activity. Its highest increase occurred in revertants originating from auxotrophs greatly predisposed to synthesize pectinases. In the case of revertants induced by mutagenes an increase in the frequency of their formation was observed, as well as an increased participation of revertants with higher pectinolytic activity compared to both their initial auxotrophic and prototrophic strain.