Compensatory evolution in diploid populations

Compensatory mutations are individually deleterious but harmless in appropriate combinations either at more than two sites within a gene or on separate genes. Considering that dominance effects of selection and heterodimer formation of gene products may affect the rate of compensatory evolution, we investigate compensatory neutral mutation models for diploid populations. Our theoretical analysis on the average time until fixation of compensatory mutations shows that these factors play an important role in reducing the fixation time of compensatory mutations if mutation rates are not low. Compensatory evolution of heterodimers is shown to occur more easily if the deleterious effects of single mutants are recessive.     

Positive selection for loss of RpoS function in Escherichia coli

Though RpoS, an alternative sigma factor, is required for survival and adaptation of Escherichia coli under stress conditions, many strains have acquired independent mutations in the rpoS gene. The reasons for this apparent selective loss and the nature of the selective agent are not well understood. In this study, we found that some wild type strains grow poorly in succinate minimal media compared with isogenic strains carrying defined RpoS null mutations. Using an rpoS+ strain harboring an operon lacZ fusion to the highly-RpoS dependent osmY promoter as an indicator strain, we tested if this differential growth characteristic could be used to selectively isolate mutants that have lost RpoS function. All isolated (Suc+) mutants exhibited attenuated beta-galactosidase expression on indicator media suggesting a loss in either RpoS or osmY promoter function. Because all Suc+ mutants were also defective in catalase activity, an OsmY-independent, RpoS-regulated function, it was likely that RpoS activity was affected. To confirm this, we sequenced PCR-amplified products containing the rpoS gene from 20 independent mutants using chromosomal DNA as a template. Sequencing and alignment analyses confirmed that all isolated mutants possessed mutated alleles of the rpoS gene. Types of mutations detected included single or multiple base deletions, insertions, and transversions. No transition mutations were identified. All identified point mutations could, under selection for restoration of beta-galactosidase, revert to rpoS+. Revertible mutation of the rpoS gene can thus function as a genetic switch that controls expression of the regulon at the population level. These results may also help to explain why independent laboratory strains have acquired mutations in this important regulatory gene.     

Isolation and Characterization of Conditional-Lethal Mutations in the Tub1 α-Tubulin Gene of the Yeast Saccharomyces Cerevisiae

Microtubules in yeast are functional components of the mitotic and meiotic spindles and are essential for nuclear movement during cell division and mating. We have isolated 70 conditional-lethal mutations in the TUB1 alpha-tubulin gene of the yeast Saccharomyces cerevisiae using a plasmid replacement technique. Of the 70 mutations isolated, 67 resulted in cold-sensitivity, one resulted in temperature-sensitivity, and two resulted in both. Fine-structure mapping revealed that the mutations were located throughout the TUB1 gene. We characterized the phenotypes caused by 38 of the mutations after shifts of mutants to the nonpermissive temperature. Populations of temperature-shifted mutant cells contained an excess of large-budded cells with undivided nuclei, consistent with the previously determined role of microtubules in yeast mitosis. Several of the mutants arrested growth with a sufficiently uniform morphology to indicate that TUB1 has at least one specific role in the progression of the yeast cell cycle. A number of the mutants had gross defects in microtubule assembly at the restrictive temperature, some with no microtubules and some with excess microtubules. Other mutants contained disorganized microtubules and nuclei. There were no obvious correlations between these phenotypes and the map positions of the mutations. Greater than 90% of the mutants examined were hypersensitive to the antimicrotubule drug benomyl. Mutations that suppressed the cold-sensitive phenotypes of two of the TUB1 alleles occurred in TUB2, the single structural gene specifying beta-tubulin.     

Novel non-suppressing mutants of Escherichia coli tRNATyr su+3.

Several addition and deletion mutations were constructed in the region of the gene for Escherichia coli tRNATyr su+3 corresponding to the dihydrouracil loop of the mature tRNA. None of these resulting mutants had detectable suppressor function compared to the parent gene yet some directed the synthesis of mature tRNA. These latter mutants may affect the ability of the tRNA to be aminoacylated or to interact with the translational machinery on the ribosome.     

An RNAi strategy for treatment of amyotrophic lateral sclerosis caused by mutant Cu,Zn superoxide dismutase

Amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) is a neurodegenerative disease characterized by motor neuron degeneration, paralysis and death. One cause of this disease is mutations in the Cu,Zn superoxide dismutase (SOD1) gene. As mutant SOD1 acquires a toxic property that kills motor neurons, by reducing the mutant protein the disease progression may be slowed or prevented. While mutant SOD1 is toxic, the wild-type SOD1 is indispensable for motor neuron health. Therefore, the ideal therapeutic strategy would be to inhibit selectively the mutant protein expression. Previously we have demonstrated that RNA interference (RNAi) can selectively inhibit some mutant SOD1 expression. However, more than 100 SOD1 mutants can cause ALS and all mutants cannot be inhibited selectively by RNAi. To overcome this obstacle, we have designed a replacement RNAi strategy. Using this strategy, all mutants and wild-type genes are inhibited by RNAi. The wild-type SOD1 function is then replaced by designed wild-type SOD1 genes that are resistant to the RNAi. Here we demonstrate the concept of this strategy.     

The molecular nature of mutants induced by X rays is altered by the presence of the radioprotector cysteamine

The molecular structure of mutants induced in human lymphoblast cells by 500 cGy X rays in the presence of the radioprotector cysteamine (25 mM) has been compared with that induced by an equally mutagenic treatment of 150 cGy X rays alone. Sets of mutants at the hypoxanthine-guanine phosphoribosyl transferase locus were analyzed by Southern blot. Of 24 mutants induced by X rays in the presence of cysteamine, 67% exhibited no change in the restriction fragment pattern and thus were defined as point mutations; 8% appeared to be total gene deletions and 25% were partial deletions or rearrangements. In contrast, among 28 mutants induced by X rays alone (Liber et al., Mutat. Res. 178, 143-153 (1987)), 46% were point mutations, while 50% were total gene deletions and only 1 mutant (4%) was a partial deletion or rearrangement. Thus mutants isolated in the presence of cysteamine consisted of more point mutations and partial deletions/rearrangements, and considerably fewer total gene deletions. These results suggest that cysteamine may protect selectively against processes which lead to large-scale molecular changes.     

Estimation of Mutation Rates Based on the Analysis of Polypeptide Constituents of Cultured Human Lymphoblastoid Cells

A subclone of a human diploid lymphoblastoid cell line, TK-6, with consistently high cloning efficiency has been used to estimate the rates of somatic mutations on the basis of protein variation detected by two-dimensional polyacrylamide gel electrophoresis. A panel of 267 polypeptide spots per gel was screened, representing the products of approximately 263 unselected loci. The rate of human somatic mutation in vitro was estimated by measuring the proportion of protein variants among cell clones isolated at various times during continuous exponential growth of a TK-6 cell population. Three mutants of spontaneous origin were observed, giving an estimated spontaneous rate of 6 x 10(-8) electrophoretic mutations per allele per cell generation (i.e., 1.2 x 10(-7) per locus per cell generation). Following treatment of cells with N-ethyl-N-nitrosourea, a total of 74 confirmed variants at 54 loci were identified among 1143 clones analyzed (approximately 601,000 allele tests). The induced variants include 65 electromorphs which exhibit altered isoelectric charge and/or apparent molecular weight and nine nullimorphs for each of which a gene product was not detected at its usual location on the gel. The induced frequency for these 65 structural gene mutants is 1.1 x 10(-4) per allele. An excess of structural gene mutations at ten known polymorphic loci and repeat mutations at these and other loci suggest nonrandomness of mutation in human somatic cells. Nullimorphs occurring at three heterozygous loci in TK-6 cells may be caused by genetic processes other than structural gene mutation.     

Direct detection of deletion mutations in the yeast DEL assay using quantitative PCR (TaqMan)

Established mutagenesis assays measure mutant frequencies at selectable loci. These assays work by encouraging the growth of mutants to form visible colonies while suppressing the growth of non-mutants. An alternative strategy is to detect DNA alterations directly. We present an example of this latter strategy using TaqMan to quantify deletion mutants in mixed cultures of Saccharomyces cerevisiae strain RS112. The RS112 strain contains two heteroallelic his3 sequences that share approximately 400bp homology and are separated by approximately 7 kb of plasmid DNA. Spontaneous and chemical-induced strand breaks that occur in this region are repaired by intrachromosomal recombination, resulting in the loss of the plasmid DNA and creation of a His prototroph. Ordinarily, these prototrophs are detected by growth on His- medium over 2-3 days. In this case, we used TaqMan to selectively detect the DNA of deletion mutants in the presence of a large excess of DNA from non-mutants. This was accomplished using primers whose annealing sites were outside the region of DNA lost due to recombination. Thus, the primers were too far apart to produce PCR products using DNA from non-mutants, but produced a robust TaqMan signal using DNA from deletion mutants. Spontaneous and chemical-induced recombination frequencies (RF) were measured in a series of time-course and dose-response experiments with direct-acting mutagens. Interestingly, chemical-induced increases in RF were observed within a few hours of initiation of exposure, demonstrating that deletion mutations in RS112 can be fixed soon after DNA damage occurs. The ability to measure RF at any time during treatment will be useful for additional mechanistic studies. Chemical-induced increases in RF were also observed in the absence of selective growth conditions. As such, detection of deletion mutations with TaqMan may be applicable to measurements of RFs at non-selectable loci in yeast and other species. Finally, chemical-induced RFs after 17 h exposure were similar to those observed after 3 days growth on selective medium. The TaqMan assay may therefore be used to screen compounds more quickly for their ability to cause deletion mutations than is currently done by plating on selective medium.     

Compensatory adaptation to the deleterious effect of antibiotic resistance in Salmonella typhimurium

Most chromosomal mutations that cause antibiotic resistance impose fitness costs on the bacteria. This biological cost can often be reduced by compensatory mutations. In Salmonella typhimurium, the nucleotide substitution AAA42 --> AAC in the rpsL gene confers resistance to streptomycin. The resulting amino acid substitution (K42N) in ribosomal protein S12 causes an increased rate of ribosomal proofreading and, as a result, the rate of protein synthesis, bacterial growth and virulence are decreased. Eighty-one independent lineages of the low-fitness, K42N mutant were evolved in the absence of antibiotic to ameliorate the costs. From the rate of fixation of compensated mutants and their fitness, the rate of compensatory mutations was estimated to be > or = 10-7 per cell per generation. The size of the population bottleneck during evolution affected fitness of the adapted mutants: a larger bottleneck resulted in higher average fitness. Only four of the evolved lineages contained streptomycin-sensitive revertants. The remaining 77 lineages contained mutants that were still fully streptomycin resistant, had retained the original resistance mutation and also acquired compensatory mutations. Most of the compensatory mutations, resulting in at least 35 different amino acid substitutions, were novel single-nucleotide substitutions in the rpsD, rpsE, rpsL or rplS genes encoding the ribosomal proteins S4, S5, S12 and L19 respectively. Our results show that the deleterious effects of a resistance mutation can be compensated by an unexpected variety of mutations.     

A neutral theory predicts multigenic aging and increased concentrations of deleterious mutations on the mitochondrial and Y chromosomes

Population genetic forces have molded the constitution of the human genome over evolutionary time, and some of the most important parameters are the initial frequency of the allele, p, the effective population size, Ne, and the selection coefficient, s. There is considerable agreement among evolutionary gerontologists that the amplitude of -s is small for alleles that are Deleterious In Late Life (DILL), and thus DILL traits are effectively neutral and should be fixed in the human population in relationship to Ne and p. Even higher rates of fixation of deleterious mutations are predicted to occur in the two nonrecombinant genomes in humans, i.e., the Y chromosome and the mitochondrial genome, as a consequence of their lower Ne than autosomes, and the predicted higher rate of fixation of deleterious alleles on the Y may explain the reduced average life span of males vs. females. The high probability of fixation of neutral and mildly deleterious mutations in the mitochondrial genome explains in part its fast rate of evolution, the high observed frequency of mitochondrial disease in relationship to this genome's small size, and may be the underlying reason for the transfer of mitochondrial genes over evolutionary time to the nucleus. The predicted higher concentration of deleterious mutations on the mitochondrial genome could have some leverage to cause more dysfunction than that predicted by mitochondrial gene number alone, because of the essential role of mitochondrial gene function in multisubunit complexes, the coupling of mitochondrial functions, the observation that some mtDNA sequences facilitate somatic mutation, and the likelihood of deleterious mutations either increasing the production of or the sensitivity to mitochondrial ROS.     

Two-Step Resistance by ESCHERICHIA COLI B to Bacteriophage T2

Numerous authors have noted the difficulty in obtaining mutants of E. coli B that are resistant to bacteriophage T2 using standard procedures of plating large numbers of cells in the presence of excess phage. Yet, T2-resistant mutants appear in continuous culture at rates in consistent with this difficulty. This paradoxical result derives from the fact that resistance to T2 usually arises as a consequence of two nonindependent mutations. Mutant bacteria resistant to phage T4 are very common and increase rapidly in continuous culture with phage T2 owing to an approximate halving of the rate at which T2 adsorbs to and kills these partially resistant mutants. The rate at which these partially resistant mutants then give rise to fully resistant mutants is approximately two orders of magnitude higher than the rate obtained by direct selection. These results are consistent with biochemical evidence that T2 adsorption to E. coli B involves both the bacterial lipopolysaccharide (to which phage T4 adsorbs) and a bacterial surface protein. However, this genetic evidence suggests that T2 can adsorb to either receptor type alone, whereas the biochemical evidence suggests that T2 requires a complex of the two receptors for adsorption to E. coli B. These results also indicate that the effects of genetic background can influence not only the selective advantage associated with particular mutations but also the rate at which certain selectively defined characteristics arise via mutation.     

Mutations affecting starch synthase III in Arabidopsis alter leaf starch structure and increase the rate of starch synthesis

The role of starch synthase (SS) III (SSIII) in the synthesis of transient starch in Arabidopsis (Arabidopsis thaliana) was investigated by characterizing the effects of two insertion mutations at the AtSS3 gene locus. Both mutations, termed Atss3-1 and Atss3-2, condition complete loss of SSIII activity and prevent normal gene expression at both the mRNA and protein levels. The mutations cause a starch excess phenotype in leaves during the light period of the growth cycle due to an apparent increase in the rate of starch synthesis. In addition, both mutations alter the physical structure of leaf starch. Significant increases were noted in the mutants in the frequency of linear chains in amylopectin with a degree of polymerization greater than approximately 60, and relatively small changes were observed in chains of degree of polymerization 4 to 50. Furthermore, starch in the Atss3-1 and Atss3-2 mutants has a higher phosphate content, approximately two times that of wild-type leaf starch. Total SS activity is increased in both Atss3 mutants and a specific SS activity appears to be up-regulated. The data indicate that, in addition to its expected direct role in starch assembly, SSIII also has a negative regulatory function in the biosynthesis of transient starch in Arabidopsis.     

Multiple mechanisms to ameliorate the fitness burden of mupirocin resistance in Salmonella typhimurium

We examined how the fitness costs of mupirocin resistance caused by mutations in the chromosomal isoleucyl-tRNA synthetase gene (ileS) can be ameliorated. Mupirocin-resistant mutants were isolated and four different, resistance-conferring point mutations in the chromosomal ileS gene were identified. Fifty independent lineages of the low-fitness, resistant mutants were serially passaged to evolve compensated mutants with increased fitness. In 34/50 of the evolved lineages, the increase in fitness resulted from additional point mutations in isoleucine tRNA synthetase (IleRS). Measurements in vitro of the kinetics of aminoacylation of wild-type and mutant enzymes showed that resistant IleRS had a reduced rate of aminoacylation due to altered interactions with both tRNAIle and ATP. The intragenic compensatory mutations improved IleRS kinetics towards the wild-type enzyme, thereby restoring bacterial fitness. Seven of the 16 lineages that lacked second-site compensatory mutations in ileS, showed an increase in ileS gene dosage, suggesting that an increased level of defective IleRS compensate for the decrease in aminoacylation activity. Our findings show that the fitness costs of ileS mutations conferring mupirocin resistance can be reduced by several types of mechanisms that may contribute to the stability of mupirocin resistance in clinical settings.     

Characterizing rice lesion mimic mutants and identifying a mutant with broad-spectrum resistance to rice blast and bacterial blight

Many plant mutants develop spontaneous lesions that resemble disease symptoms in the absence of pathogen attack. In several pathosystems, lesion mimic mutations have been shown to be involved in programmed cell death, which in some instances leads to enhanced disease resistance to multiple pathogens. We investigated the relationship between spontaneous cell death and disease resistance in rice with nine mutants with a range of lesion mimic phenotypes. All nine mutations are controlled by recessive genes and some of these mutants have stunted growth and other abnormal characteristics. The lesion mimics that appeared on the leaves of these mutants were caused by cell death as measured by trypan blue staining. Activation of six defense-related genes was observed in most of the mutants when the mimic lesions developed. Four mutants exhibited significant enhanced resistance to rice blast. One of the mutants, spl11, confers non-race-specific resistance not only to blast but also to bacterial blight. The level of resistance in the spl11 mutant to the two pathogens correlates with the defense-related gene expression and lesion development on the leaves. The results suggest that some lesion mimic mutations in rice may be involved in disease resistance, and cloning of these genes may provide a clue to developing broad-spectrum resistance to diverse pathogens.     

Relationship between DNA Polymorphism and Fixation Time

When there is no recombination among nucleotide sites in DNA sequences, DNA polymorphism and fixation of mutants at nucleotide sites are mutually related. Using the method of gene genealogy, the relationship between the DNA polymorphism and the fixation of mutant nucleotide was quantitatively investigated under the assumption that mutants are selectively neutral, that there is no recombination among nucleotide sites, and that the population is a random mating population with N diploid individuals. The results obtained indicate that the expected number of nucleotide differences between two DNA sequences randomly sampled from the population is 42% less when a mutant at a particular nucleotide site reaches fixation than at a random time, and that heterozygosity is also expected to be less when fixation takes place than at a random time, but the amount of reduction depends on the value of 4Nv in this case, where v is the mutation rate per DNA sequence per generation. The formula for obtaining the expected number of nucleotide differences between the two DNA sequences for a given fixation time is also derived, and indicates that, even when it takes a large number of generations for a mutant to reach fixation, this number is 33% less than at a random time. The computer simulation conducted suggests that the expected number of nucleotide differences between the two DNA sequences at the time when an advantageous mutant becomes fixed is essentially the same as that of neutral mutant if the fixation time is the same. The effect of recombination on the amount of DNA polymorphism was also investigated by using computer simulation.     

ScoC mediates catabolite repression of sporulation in Bacillus subtilis

Sporulation in Bacillus subtilis can be triggered by carbon catabolite limitation. Conversely, carbon source excess can repress the production of extracellular enzymes, motility, and sporulation. Recent studies have implicated a pH-sensing mechanism, involving AbrB, the TCA cycle, Spo0K, and sigmaH in controlling the catabolite repression of sporulation gene expression. In an accompanying paper, we demonstrate that the AbrB-dependent pH-sensing mechanism may not be the only means by which carbon catabolites affect sporulation. In the studies reported here, we have examined the molecular basis underlying the catabolite repression phenotype of mutations in the hpr (scoC), rpoD (crsA47), and spo0A (rvtA11) loci. Loss of function mutations in hpr (scoC) restored sporulation gene expression and sporulation in the presence of excess catabolite(s), suggesting that Hpr (ScoC) has a pivotal role in mediating catabolite repression. Moreover, hpr gene expression increased substantially in the presence of excess catabolite(s), further supporting the involvement of Hpr (ScoC) in the carbon catabolite response system. We suggest that alterations in the phosphorelay response to catabolites may be one mechanism by which catabolite-resistant mutants such as crsA and rvtA are able to sporulate in the presence of excess glucose.     

secD, a new gene involved in protein export in Escherichia coli.

New mutants of Escherichia coli altered in protein export were identified in phoA-lacZ and lamB-lacZ gene fusion strains by searching for mutants that showed an altered lactose phenotype. Several mutations mapped in a new gene, secD. These mutants were, in general, cold sensitive for growth, and the mutations led to an accumulation of precursor of exported proteins. The secD gene is closely linked to tsx on the E. coli chromosome, but separable from another gene proposed to be involved in export, ssaD, which maps nearby. A plasmid carrying secD+ was identified and used to show that the mutations are recessive. The secD gene may code for a component of the cellular export machinery.     

Glucose Utilization and Ethanolic Fermentation by Wild Type and Extrachromosomal Mutants of Neurospora crassa

Logarithmic growth rates, maximal biomass, specific glucose utilization rates, and ethanol accumulation were measured in aerobic cultures of wild type and extrachromosomal mutants of Neurospora crassa. Maximal biomass and ethanol accumulation of wild type and [mi-1] were proportional to the initial glucose concentration in the range of 2 to 10%. The specific rates of glucose utilization by the mutants were 13- to 20-fold greater than those of wild type in young cultures. The specific rates of glucose utilization by wild type, however, were increased threefold by increasing the ammonium ion concentration in the preculture medium. The suppressor gene f(+) suppressed the excessive glucose utilization and enhanced the growth rate and maximal biomass of [mi-1]. When the mutants were utilizing glucose at excessive rates, ethanol did not appear in the culture medium. Ethanol accumulation was maximum at stationary phase or thereafter, but there was little difference between the maxima of the mutants and wild type. The molar efficiency of the conversion of glucose to ethanol during the entire culture period of wild type and mutants was about 50% and, in the latter stages of fermentation, approached 100%. Replacement of ammonium ion by nitrate in the culture medium suppressed ethanol accumulation by wild type. The relationship of these results to previous observations on respiratory adaptation are discussed. We suggest that the Pasteur effect, the inhibition of fermentation by respiration, may be operative in N. crassa. Factors such as nitrogen source and concentration and oxygen tension, which may serve primarily to regulate the amount and form of respiration would, therefore, indirectly regulate fermentation. The mutants, although transiently deficient in terminal respiratory activity, do not accumulate more ethanol than wild type and, therefore, apparently do not ferment in excess to obtain additional adenosine 5'-triphosphate. We suggest that the excess activity of the alternate form of respiration of the mutants may be related to their excessive rate of glucose utilization by way of the pentose phosphate pathway and the oxidation of excess reduced nicotinamide adenine dinucleotide.     

The role of pre-replication and post-replication processes in mutation induction in Haemophilus influenzae by N-methyl-N'-nitro-N-nitrosoguanidine.

Studies were carried out on the repair and fixation of premutational damage induced in Haemophilus influenzae by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The studies employed a temperature-sensitive DNA elongation mutant (dna9) and its combinations with mutants defective in pyrimidine dimer excision (uvr1, uvr2) and in recombination (rec1). The dna9 mutant is shown to be leaky, allowing about 1% of the normal rate of DNA synthesis at the restrictive temperature. Repair of premutational lesions was detected by a decline in mutation frequency with increasing delay in DNA replication in dna9 at the restrictive temperature. This repair is unaffected by the pyrimidine dimer excision system. Mutation fixation was detected by the ability of DNA from treated and then lysed cells to transfer mutants to recipient cells by transformation. Some fixation occurred at the restrictive temperature but much less than at the non-restrictive temperature suggesting that an appreciable minority of the mutations resulted from lesions introduced near the replication fork but that the majority of mutations arise from lesions introduced at some distance from the fork, perhaps randomly. The DNA synthesized immediately after MNNG treatment is of lower molecular weight than normal and returns to normal with time. This return is blocked in the rec1 mutant, suggesting that recombination is involved. The possible role of this process in MNNG mutagenesis is discussed.