There is widespread agreement that the clamp loader of the Escherichia coli replicase has the composition DnaX3δδ’χψ. Two DnaX proteins exist in E. coli, full length τ and a truncated γ that is created by ribosomal frameshifting. τ binds DNA polymerase III tightly; γ does not. There is a controversy as to whether or not DNA polymerase III holoenzyme (Pol III HE) contains γ. A three-τ form of Pol III HE would contain three Pol IIIs. Proponents of the three-τ hypothesis have claimed that γ found in Pol III HE might be a proteolysis product of τ. To resolve this controversy, we constructed a strain that expressed only τ from a mutated chromosomal dnaX. γ containing a C-terminal biotinylation tag (γ-Ctag) was provided in trans at physiological levels from a plasmid. A 2000-fold purification of Pol III* (all Pol III HE subunits except β) from this strain contained one molecule of γ-Ctag per Pol III* assembly, indicating that the dominant form of Pol III* in cells is Pol III2τ2 γδδ’χψ. Revealing a role for γ in cells, mutants that express only τ display sensitivity to ultraviolet light and reduction in DNA Pol IV-dependent mutagenesis associated with double-strand-break repair, and impaired maintenance of an F’ episome.
Bacteria, yeast and human cancer cells possess mechanisms of mutagenesis upregulated by stress responses. Stress-inducible mutagenesis potentially accelerates adaptation, and may provide important models for mutagenesis that drives cancers, host pathogen interactions, antibiotic resistance and possibly much of evolution generally. In Escherichia coli repair of double-strand breaks (DSBs) becomes mutagenic, using low-fidelity DNA polymerases under the control of the SOS DNA-damage response and RpoS general stress response, which upregulate and allow the action of error-prone DNA polymerases IV (DinB), II and V to make mutations during repair. Pol IV is implied to compete with and replace high-fidelity DNA polymerases at the DSB-repair replisome, causing mutagenesis. We report that up-regulated Pol IV is not sufficient for mutagenic break repair (MBR); damaged bases in the DNA are also required, and that in starvation-stressed cells, these are caused by reactive-oxygen species (ROS). First, MBR is reduced by either ROS-scavenging agents or constitutive activation of oxidative-damage responses, both of which reduce cellular ROS levels. The ROS promote MBR other than by causing DSBs, saturating mismatch repair, oxidizing proteins, or inducing the SOS response or the general stress response. We find that ROS drive MBR through oxidized guanines (8-oxo-dG) in DNA, in that overproduction of a glycosylase that removes 8-oxo-dG from DNA prevents MBR. Further, other damaged DNA bases can substitute for 8-oxo-dG because ROS-scavenged cells resume MBR if either DNA pyrimidine dimers or alkylated bases are induced. We hypothesize that damaged bases in DNA pause the replisome and allow the critical switch from high fidelity to error-prone DNA polymerases in the DSB-repair replisome, thus allowing MBR. The data imply that in addition to the indirect stress-response controlled switch to MBR, a direct cis-acting switch to MBR occurs independently of DNA breakage, caused by ROS oxidation of DNA potentially regulated by ROS regulators.
Summary Transgenomics is the process of introducing genomic clones from a donor species into a recipient species and then screening the resultant transgenic lines for phenotypes of interest. This method might allow us to find genes involved in the evolution of phenotypic differences between species as well as genes that have the potential to contribute to reproductive isolation: potential speciation genes.More than 1,100 ~20 Kbp genomic clones from Leavenworthia alabamica were moved into Arabidopsis thaliana by transformation. After screening a single primary transformant for each line, clones associated with mutant phenotypes were tested for repeatability and cosegregation.We found 84 clones with possible phenotypic effects of which eight were repeatedly associated with the same phenotype. One clone, 11_11B, cosegregated with a short fruit phenotype. Further study showed that 11_11B affects seed development, with as much as one-third of the seeds aborted in some fruit.Transgenomics is a viable strategy for discovering genes of evolutionary interest. We identify methods to reduce false positives and false negatives in the future. 11_11B can be viewed as a potential speciation gene, illustrating the value of transgenomics for studying the molecular basis of reproductive isolation.
Summary• Leavenworthia crassa is a rosette flowering species that differs from inflorescence flowering species, such as Arabidopsis thaliana, in having elongated pedicels and shortened interfloral internodes on the main axis. Based on previous experiments, we hypothesized that changes to the L. crassa TFL1 ortholog, LcrTFL1, were important in the evolution of rosette flowering.• We isolated LcrTFL1 and introduced a genomic construct into tfl1 mutant A. thaliana plants. We also generated and analyzed EGFP-LcrTFL1 reporter-fusion lines, and LcrTFL1 ⁄ LcrLFY doubly transgenic lines.• The transgene rescued the mutant defects, but manifested gain-of-function phenotypes. However, LcrTFL1 lines differed from 35S:TFL1 lines in several regards. Defects in floral meristem identity establishment were observed, as was the production of flowers with extra petals. We also noted features that resemble rosette flowering: LcrTFL1 lines produced significantly shorter interfloral internodes and significantly longer pedicels than either wild-type or 35S:TFL1 plants.• Our data show that there are substantive differences in the regulation and ⁄ or function of TFL1 orthologs between A. thaliana and L. crassa. These may reflect changes that occurred during the evolution of rosette flowering in Leavenworthia, but, if so, our results show that additional, as-yet-unidentified genes were involved in this instance of architectural evolution.
Mechanisms of mutation upregulated by stress responses have been described in several organisms from bacteria to human. These mechanisms might accelerate genetic change specifically when cells are maladapted to their environment. Stress-induced mutation mechanisms differ in their genetic requirements from mutation in growing cells, occurring by different mechanisms in different assay systems, but having in common a requirement for the induction of stress-responses. Here, we review progress in two areas relevant to stress-response-dependent mutagenic DNA break repair mechanisms in Escherichia coli. First, we review evidence that relates mutation to transcription. This connection might allow mutagenesis in transcribed regions, including those relevant to any stress being experienced, opening the possibility that mutations could be targeted to regions where mutation might be advantageous under conditions of a specific stress. We review the mechanisms by which replication initiated by transcription can lead to mutation. Second, we review recent findings that, although stress-induced mutation does not require exogenous DNA-damaging agents, it does require the presence of damaged bases in DNA. For starved E. coli, endogenous oxygen radicals cause these altered bases. We postulate that damaged bases stall the replisome, which, we suggest, is required for DNA-polymerase exchange, allowing the action of low-fidelity DNA polymerases that promote mutation.
Many advances in our understanding of the genetic basis of species differences have arisen from transformation experiments, which allow us to study the effect of genes from one species (the donor) when placed in the genetic background of another species (the recipient). Such interspecies transformation experiments are usually focused on candidate genes – genes that, based on work in model systems, are suspected to be responsible for certain phenotypic differences between the donor and recipient species. We suggest that the high efficiency of transformation in a few plant species, most notably Arabidopsis thaliana, combined with the small size of typical plant genes and their cis-regulatory regions allow implementation of a screening strategy that does not depend upon a priori candidate gene identification. This approach, transgenomics, entails moving many large genomic inserts of a donor species into the wild type background of a recipient species and then screening for dominant phenotypic effects. As a proof of concept, we recently conducted a transgenomic screen that analyzed more than 1100 random, large genomic inserts of the Alabama gladecress Leavenworthia alabamica for dominant phenotypic effects in the A. thaliana background. This screen identified one insert that shortens fruit and decreases A. thaliana fertility. In this paper we discuss the principles of transgenomic screens and suggest methods to help minimize the frequencies of false positive and false negative results. We argue that, because transgenomics avoids committing in advance to candidate genes it has the potential to help us identify truly novel genes or cryptic functions of known genes. Given the valuable knowledge that is likely to be gained, we believe the time is ripe for the plant evolutionary community to invest in transgenomic screens, at least in the mustard family Brassicaceae where many species are amenable to efficient transformation.
Este artigo propõe reflexões sobre o processo histórico de expansão dos direitos sociais nas sociedades modernas. Trata-se de um ensaio pautado na teoria figuracional de Norbert Elias, na qual aponta que as diferentes direções dos acontecimentos sociais ocorrem como resultantes das relações de forças existentes entre diferentes configurações sociais. Observamos que a educação, embora constitua um direito social amplamente assumido e difundido como primordial nas sociedades ocidentais visando o desenvolvimento humano, contudo vem sendo uma arena para interesses distintos. Em nossa análise há indicativos de que a apropriação pelo capitalismo de determinados elementos propostos pelos ideais socialistas contribuíram para a expansão de direitos nas sociedades modernas. As políticas neoliberais na atualidade, fundamentadas na disciplina de mercado, trazem implicações que geram tensões na efetividade de tais direitos, em especial ao direito à educação. Embora os dispositivos legais sustentem a educação como um direito voltado à formação humana em sua plenitude, diferentes interesses o conduzem, por meio de estratégias diversas, prioritariamente, para a formação de mão de obra a serviço do mercado econômico.
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