Evidence that selected amplification of a bacterial lac frameshift allele stimulates Lac(+) reversion (adaptive mutation) with or without general hypermutability

In the genetic system of Cairns and Foster, a nongrowing population of an E. coli lac frameshift mutant appears to specifically accumulate Lac(+) revertants when starved on medium including lactose (adaptive mutation). This behavior has been attributed to stress-induced general mutagenesis in a subpopulation of starved cells (the hypermutable state model). We have suggested that, on the contrary, stress has no direct effect on mutability but favors only growth of cells that amplify their leaky mutant lac region (the amplification mutagenesis model). Selection enhances reversion primarily by increasing the mutant lac copy number within each developing clone on the selection plate. The observed general mutagenesis is attributed to a side effect of growth with an amplification-induction of SOS by DNA fragments released from a tandem array of lac copies. Here we show that the S. enterica version of the Cairns system shows SOS-dependent general mutagenesis and behaves in every way like the original E. coli system. In both systems, lac revertants are mutagenized during selection. Eliminating the 35-fold increase in mutation rate reduces revertant number only 2- to 4-fold. This discrepancy is due to continued growth of amplification cells until some clones manage to revert without mutagenesis solely by increasing their lac copy number. Reversion in the absence of mutagenesis is still dependent on RecA function, as expected if it depends on lac amplification (a recombination-dependent process). These observations support the amplification mutagenesis model.     

Conjugation is not required for adaptive reversion of an episomal frameshift mutation in Escherichia coli.

Adaptive reversion of a lac allele on an F' episome in a strain of Escherichia coli is dependent on the RecA-BCD pathway for recombination and is enhanced by conjugal functions. However, conjugation, i.e., transfer of the episome, whether between distinct populations of cells or between newly divided siblings, does not contribute to the mutational process.     

A second transthyretin mutation at position 33 (Leu/Phe) associated with familial amyloidotic polyneuropathy

Genomic DNA was isolated from peripheral blood lymphocytes of a patient with familial amyloidotic polyneuropathy (FAP) and the transthyretin (TTR) gene examined for sequence mutations. Polymerase chain reaction was used to asymmetrically amplify the TTR exons. Direct DNA sequencing of the PCR product revealed a C for T mutation at the first base of codon 33 located in exon 2 of one transthyretin gene. This resulted in a substitution of leucine for phenylalanine at position 33. Exons 3 and 4 were examined and found to be normal. The mutation creates a novel DdeI restriction site at the point of the mutation.     

The effect of genomic information on optimal contribution selection in livestock breeding programs

Background

Long-term benefits in animal breeding programs require that increases in genetic merit be balanced with the need to maintain diversity (lost due to inbreeding). This can be achieved by using optimal contribution selection. The availability of high-density DNA marker information enables the incorporation of genomic data into optimal contribution selection but this raises the question about how this information affects the balance between genetic merit and diversity.

Methods

The effect of using genomic information in optimal contribution selection was examined based on simulated and real data on dairy bulls. We compared the genetic merit of selected animals at various levels of co-ancestry restrictions when using estimated breeding values based on parent average, genomic or progeny test information. Furthermore, we estimated the proportion of variation in estimated breeding values that is due to within-family differences.

Results

Optimal selection on genomic estimated breeding values increased genetic gain. Genetic merit was further increased using genomic rather than pedigree-based measures of co-ancestry under an inbreeding restriction policy. Using genomic instead of pedigree relationships to restrict inbreeding had a significant effect only when the population consisted of many large full-sib families; with a half-sib family structure, no difference was observed. In real data from dairy bulls, optimal contribution selection based on genomic estimated breeding values allowed for additional improvements in genetic merit at low to moderate inbreeding levels. Genomic estimated breeding values were more accurate and showed more within-family variation than parent average breeding values; for genomic estimated breeding values, 30 to 40% of the variation was due to within-family differences. Finally, there was no difference between constraining inbreeding via pedigree or genomic relationships in the real data.

Conclusions

The use of genomic estimated breeding values increased genetic gain in optimal contribution selection. Genomic estimated breeding values were more accurate and showed more within-family variation, which led to higher genetic gains for the same restriction on inbreeding. Using genomic relationships to restrict inbreeding provided no additional gain, except in the case of very large full-sib families.     

Beneficial mutation selection balance and the effect of linkage on positive selection

When beneficial mutations are rare, they accumulate by a series of selective sweeps. But when they are common, many beneficial mutations will occur before any can fix, so there will be many different mutant lineages in the population concurrently. In an asexual population, these different mutant lineages interfere and not all can fix simultaneously. In addition, further beneficial mutations can accumulate in mutant lineages while these are still a minority of the population. In this article, we analyze the dynamics of such multiple mutations and the interplay between multiple mutations and interference between clones. These result in substantial variation in fitness accumulating within a single asexual population. The amount of variation is determined by a balance between selection, which destroys variation, and beneficial mutations, which create more. The behavior depends in a subtle way on the population parameters: the population size, the beneficial mutation rate, and the distribution of the fitness increments of the potential beneficial mutations. The mutation-selection balance leads to a continually evolving population with a steady-state fitness variation. This variation increases logarithmically with both population size and mutation rate and sets the rate at which the population accumulates beneficial mutations, which thus also grows only logarithmically with population size and mutation rate. These results imply that mutator phenotypes are less effective in larger asexual populations. They also have consequences for the advantages (or disadvantages) of sex via the Fisher-Muller effect; these are discussed briefly.     

The impact of epistatic selection on the genomic traces of selection

The rapid accumulation of genomic data has led to an explosion of studies searching for signals of past selection left within DNA sequences. Yet the majority of theoretical studies investigating the traces of selection have assumed a simple form of selection, without interactions among selectively fixed sites. Fitness interactions—‘epistasis’—are commonplace, however, and take on a myriad of forms ( Whitlock et al. 1995 ; Segrèet al. 2005 ; Phillips 2008 ). It is thus important to determine how such epistasis would influence selective sweeps. On p. 5018 of this issue, Takahasi (2009) explores the effect of epistasis on genetic variation neighbouring two sites that interact in determining fitness, finding that such epistasis has a dramatic impact on the genetic variability in regions surrounding the interacting sites.     

The 3-methylcholanthrene-inducible UDP-glucuronosyltransferase deficiency in the hyperbilirubinemic rat (Gunn rat) is caused by a -1 frameshift mutation

The Gunn rat is a mutant strain of Wistar rat which has unconjugated hyperbilirubinemia as a result of the absence of hepatic UDP-glucuronosyltransferase (UDPGT) activity toward bilirubin. The Gunn rat is also deficient in a 3-methylcholanthrene (MC)-inducible UDPGT isoenzyme that has high activity toward phenolic substrates. We have isolated and sequenced a cDNA, designated 4-NP UDPGT, which encodes an MC-inducible UDPGT from normal Wistar rat livers (Iyanagi, T., Haniu, M., Sogawa, F., Fujii-Kuriyama, Y., Watanabe, S., Shively, J.E., and Anan, K.F. (1986) J. Biol. Chem. 261, 15607-15614). In the present study, we found that this cDNA detected MC-inducible UDPGT mRNA in the MC-treated homozygous Gunn rat liver. The level of this mRNA, however, was significantly lower than that of normal Wistar livers. The size of mRNA in Gunn rats was identical to that of the functionally mature UDPGT mRNA in Wistar rats, but the MC-inducible UDPGT protein was absent from homozygous Gunn rat microsomes. We therefore made a cDNA library from MC-treated Gunn rat liver mRNA and isolated cDNA clones, using the 4-NP UDPGT cDNA as a probe. Sequencing analysis of these cDNA clones revealed a single base deletion in the coding region. Northern blot analysis of mRNAs from normal Wistar and heterozygous and homozygous Gunn rats livers was performed using specific oligonucleotide probes, and the results confirmed the presence of mRNA containing the single base deletion in heterozygous and homozygous Gunn rats. These data suggested that the defect of the MC-inducible isoenzyme in Gunn rats arises from a -1 frameshift mutation that removes 115 amino acids from the COOH terminus.     

Properties of adaptive walks on uncorrelated landscapes under strong selection and weak mutation

We examine properties of adaptive walks on uncorrelated (i.e. random) fitness landscapes starting from moderately fit genotypes under strong selection weak mutation. As an extension of Orr's model for a single step in an adaptive walk under these conditions, we show that the fitness rank of the dominant genotype in a population after the fixation of a beneficial mutation is, on average, (i+6)/4, where i is the fitness rank of the starting genotype. This accounts for the change in rank due to acquiring a new set of single-mutation neighbors after fixing a new allele through natural selection. Under this scenario, adaptive walks can be modeled as a simple Markov chain on the space of possible fitness ranks with an absorbing state at i = 1, from which no beneficial mutations are accessible. We find that these walks are typically short and are often completed in a single step when starting from a moderately fit genotype. As in Orr's original model, these results are insensitive to both the distribution of fitness effects and most biological details of the system under consideration.     

Restriction fragment analysis confirms the position 33 mutation in transthyretin from an Israeli patient (SKO) with familial amyloidotic polyneuropathy

Transthyretin isolated from amyloid fibrils from an Israeli patient with Familial Amyloidotic Polyneuropathy was sequenced by two research groups. One laboratory reported a position 49 Thr----Gly substitution, while the other noted a Phe for Ile interchange at amino acid 33. We used a transthyretin cDNA probe to study DNA from this patient by Southern blotting. The DNA displayed the unique Bcl I restriction site predicted by the mutation in codon 33. Because of the close size of the normal (6.40 kb), and variant (6.27 kb) fragments, the variant was more easily demonstrated after digestion with both Bcl I and Sph I, which generated two easily resolvable fragments of 2.39 and 2.27 kb.     

JADE: a distributed Java application for deleterious genomic mutation (DGM) estimation

SUMMARY: The characterization of deleterious genomic mutation (DGM) is of central significance for evolutionary biology and genetic studies. Fitness moment method has been developed to efficiently characterize DGM from natural population directly. In order to enable researchers to employ this method for theoretical and empirical research on characterizing DGM, we here present a distributed Java Application for DGM Estimation (JADE). AVAILABILITY: http://orclinux.creighton.edu/DGM/index.htm.     

Response to genomic selection: The Bulmer effect and the potential of genomic selection when the number of phenotypic records is limiting

Background

Over the last ten years, genomic selection has developed enormously. Simulations and results on real data suggest that breeding values can be predicted with high accuracy using genetic markers alone. However, to reach high accuracies, large reference populations are needed. In many livestock populations or even species, such populations cannot be established when traits are difficult or expensive to record, or when the population size is small. The value of genomic selection is then questionable.

Methods

In this study, we compare traditional breeding schemes based on own performance or progeny information to genomic selection schemes, for which the number of phenotypic records is limiting. Deterministic simulations were performed using selection index theory. Our focus was on the equilibrium response obtained after a few generations of selection. Therefore, we first investigated the magnitude of the Bulmer effect with genomic selection.

Results

Results showed that the reduction in response due to the Bulmer effect is the same for genomic selection as for selection based on traditional BLUP estimated breeding values, and is independent of the accuracy of selection. The reduction in response with genomic selection is greater than with selection based directly on phenotypes without the use of pedigree information, such as mass selection. To maximize the accuracy of genomic estimated breeding values when the number of phenotypic records is limiting, the same individuals should be phenotyped and genotyped, rather than genotyping parents and phenotyping their progeny. When the generation interval cannot be reduced with genomic selection, large reference populations are required to obtain a similar response to that with selection based on BLUP estimated breeding values based on own performance or progeny information. However, when a genomic selection scheme has a moderate decrease in generation interval, relatively small reference population sizes are needed to obtain a similar response to that with selection on traditional BLUP estimated breeding values.

Conclusions

When the trait of interest cannot be recorded on the selection candidate, genomic selection schemes are very attractive even when the number of phenotypic records is limited, because traditional breeding requires progeny testing schemes with long generation intervals in those cases.     

The processing of a Benzo(a)pyrene adduct into a frameshift or a base substitution mutation requires a different set of genes in Escherichia coli

Replication through a single DNA lesion may give rise to a panel of translesion synthesis (TLS) events, which comprise error-free TLS, base substitutions and frameshift mutations. In order to determine the genetic control of the various TLS events induced by a single lesion, we have chosen the major N2-dG adduct of (+)-anti-Benzo(a)pyrene diol epoxide [(+)-anti-BPDE] adduct located within a short run of guanines as a model lesion. Within this sequence context, in addition to the major event, i.e. error-free TLS, the adduct also induces base substitutions (mostly G --> T transversions) and -1 frameshift mutations. The pathway leading to G --> T base substitution mutagenesis appears to be SOS independent, suggesting that TLS is most probably performed by the replicative Pol III holoenzyme itself. In contrast, both error-free and frameshift TLS pathways are dependent upon SOS-encoded functions that belong to the pool of inducible DNA polymerases specialized in TLS (translesional DNA polymerases), namely umuDC (Pol V) and dinB (Pol IV). It is likely that, given the diversity of conformations that can be adopted by lesion-containing replication intermediates, cells use one or several translesional DNA polymerases to achieve TLS.     

The effect of two chemical mutagens ENU and MMS on MR-mediated reversion of an insertion-sequence mutation in Drosophila melanogaster

The effects of two mutagens ENU and MMS characterized by different alkylation patterns have been studied on the reversion of an MR-induced singed mutation to wild-type. Reversion of this unstable singed mutation under the influence of MR is assumed to represent the removal or transposition of an insertion element. Since MR acts primarily in spermatogonia, the mutagens were fed to 1st instar larvae. Recessive lethal tests were carried out simultaneously to calibrate for the mutagenic effectiveness of the chemicals. For both powerful mutagens, it was observed that the frequency of reversion remained far below of what would have been expected on the basis of the mutagenic effectiveness, as registered in the lethal tests. Thus 1 mM ENU, 5 mM and 10 mM MMS did not affect the reversion frequency at all, and with 3 mM ENU only a doubling of the reversion frequency was observed, despite a 5-fold increase in the lethal frequency. The threshold at 1 mM EMU and the low effectiveness of 3 mM on the reversion process are taken as an indication that ENU affected the transposition process in an indirect manner, rather than the excision events themselves. The data obtained with Drosophila are consistent with the microbial observations in that mutation involving removal or transposition of an insertion element is not affected by mutagenic treatments. This finding may have consequences for the evaluation of induced genetic damage on the basis of the spontaneous load of genetic detriment in man.

An incidental observation was that non-MR Cy larvae exhibited greater sensitivity to the induction of recessive lethals by MMS than MR-individuals.     

The right end of MudI(Ap,lac)

Stable derivatives of the bacteriophage MudI(Ap,lac) were used to generate operon fusions in S. typhimurium which exhibit a sectoring phenotype with respect to lacZ expression. The Lac^- to Lac⁺ conversion was shown to be the result of small deletions involving the right end of the MudI element. DNA sequence analysis of several different fusions revealed that this end of MudI(Ap,lac) contains an assymetric inverted repeat of the attR site found in the wild-type Mu phage. A model is presented which explains how such a structure was formed in the construction of MudI(Ap,lac). In addition, this model explains the observed deletion formation and the Lac^- to Lac⁺ conversion in the sectoring fusions.This paper is dedicated to our padrinos, John and Marge Ingraham, whose love of truth has served us as constant inspiration