Application of the theory of planned behaviour to career choice: The role of an improved measure of emotion

Adherents of the Theory of Planned Behaviour (TPB) propose that intention to perform behaviour can be predicted by attitudes, subjective norms and perceived behavioural control. Recent studies, however, have indicated that the standard TPB predictor variables account for 28% to 40% of the variance in intention, leaving a considerable percentage of the variance in intentions to be explained. Attitude is traditionally measured by the valence associated with the intention. The present study employed an improved measure of attitude, including both emotional dimensions of valence and arousal (Bradley & Lang, 1999), rather than using valence alone, and tested whether this enhanced measure increased the prediction of career choice in 140 university students. It was hypothesized that using both valence and arousal, to operationalise attitude, should account for more of the variance associated with intended career choice, rather than using valence alone. Consistent with the hypothesis, a logistic regression analysis revealed that the inclusion of arousal accounted for an additional 9% of the variance in intention to become a professional in the discipline studied. It may therefore be possible to increase the amount of explained variance using the TPB by including arousal in operationalising attitude as a predictor of behavioural intention

Mutability and Importance of a Hypermutable Cell Subpopulation that Produces Stress-Induced Mutants in Escherichia coli

In bacterial, yeast, and human cells, stress-induced mutation mechanisms are induced in growth-limiting environments and produce non-adaptive and adaptive mutations. These mechanisms may accelerate evolution specifically when cells are maladapted to their environments, i.e., when they are are stressed. One mechanism of stress-induced mutagenesis in Escherichia coli occurs by error-prone DNA double-strand break (DSB) repair. This mechanism was linked previously to a differentiated subpopulation of cells with a transiently elevated mutation rate, a hypermutable cell subpopulation (HMS). The HMS could be important, producing essentially all stress-induced mutants. Alternatively, the HMS was proposed to produce only a minority of stress-induced mutants, i.e., it was proposed to be peripheral. We characterize three aspects of the HMS. First, using improved mutation-detection methods, we estimate the number of mutations per genome of HMS-derived cells and find that it is compatible with fitness after the HMS state. This implies that these mutants are not necessarily an evolutionary dead end, and could contribute to adaptive evolution. Second, we show that stress-induced Lac+ mutants, with and without evidence of descent from the HMS, have similar Lac+ mutation sequences. This provides evidence that HMS-descended and most stress-induced mutants form via a common mechanism. Third, mutation-stimulating DSBs introduced via I-SceI endonuclease in vivo do not promote Lac+ mutation independently of the HMS. This and the previous finding support the hypothesis that the HMS underlies most stress-induced mutants, not just a minority of them, i.e., it is important. We consider a model in which HMS differentiation is controlled by stress responses. Differentiation of an HMS potentially limits the risks of mutagenesis in cell clones

Spetsialiseerunud DNA polümeraaside roll Pseudomonas putida rakkudes toimuvates mutatsiooni protsessides

Bacteria possess an amazing ability to adapt extremely diverse environments. Generation of mutations is an important source of biological diversity and evolution therefore contributing to adaptibility. Several lines of evidence indicate that there is positive correlation between stressful growth-restrictive environmental conditions and genetic variability within bacterial populations. Among different processes, error-prone DNA synthesis by specialized DNA polymerases has been shown to contribute to stress-induced mutagenesis in stationary-phase bacteria. Literature review of this thesis is focused on characterization of specialized DNA polymerases, their biological functions and involvement in stress-induced mutagenesis. In experimental part of my thesis I describe the involvement of specialized DNA polymerases in mutagenic processes in Pseudomonas putida stationary-phase population. P. putida is a soil bacterium colonizing very diverse habitats, being therefore continuously exposed to various environmental stresses including nutrient limitation. In order to investigate mutational processes in P. putida we constructed a set of novel test systems that allowing monitoring of 1-bp deletions and different base substitution mutations in separately throughout long-term period (at least 2 weeks) of carbon source starvation. The mutagenesis experiments with P. putida strains deficient for different specialized DNA polymerases revealed following: 1.Generation 1-bp frameshift mutations is significantly elevated as carbon source limitation proceeds more than one week. The occurrence of 1-bp deletions in late stationary-phase cells of P. putida depends on the presence of a Y-family DNA polymerase IV. 2.The DNA polymerase DnaE2, a homologue of -subunit of replicative DNA polymerase III and an Y-family DNA polymerase ImuB of P. putida that is encoded by DNA damage-inducible multiple gene cassette, have opposite effects on mutagenesis. ImuB contributes to the generation of both base substitution mutations and 1-bp deletions in P. putida stationary phase population whereas P. putida DnaE2 has a reducing effect on the occurrence of base substitutions which is in strict contrast with previously studied DnaE2 homologues of other organisms. 3.A DNA polymerase V homologue encoded by the rulAB genes of the toluene degradation plasmid pWW0 increases the probability of P. putida cells to accumulate beneficial mutations permitting to gain the growth advantage in stationary phase population. Bakteritele on üldiselt iseloomulik võime nii füsioloogiliselt kui ka geneetiliselt kiiresti muutunud keskkonnatingimustega kohaneda. Geneetilise adapteerumise ja evolutsioneerumise aluseks on erinevad mutatsiooniprotsessid. Ebasobivad, bakterite kasvu pärssivad keskkonnatingimused soodustavad mutatsioonide tekkimist. Mutatsioonide tekkes on oluline roll spetsialiseerunud DNA polümeraaside poolt läbiviidaval vigaderohkel DNA sünteesil. Käesoleva töö kirjanduse ülevaates kirjeldan erinevaid bakteriaalseid spetsialiseerunud DNA polümeraase ja nende funktsioone mutatsiooniprotsessides, sealhulgas ka stressi poolt indutseeritud mutageneesis. Doktoritöös esitatud tulemused käsitlevad erinevate spetsialiseerunud DNA polümeraaside osalust Pseudomonas putida nälgivates rakkudes toimuvates mutatsiooni protsessides. P. putida on mullabakter, mis asustab erinevaid vee ja pinnase keskkondi. Mullabakterite looduslikus elukeskkonnas on pikki perioode kestev toiteainete puudus tavaline. Pikaajalise süsinikunälja tingimustes viibivas bakteripopulatsioonis toimuvate mutatsiooniprotsesside uurimiseks konstrueerisime komplekti uusi, fenooli süsinikuallikana kasutuselevõtul põhinevaid testsüsteeme, mis võimaldavad jälgida eraldi nii asendusmutatsioonide kui ka ühenukleotiidsete deletsioonide teket pseudomonaadide populatsioonis pikaajalise süsinikunälja tingimustes. Erinevate spetsialiseerunud DNA polümeraaside suhtes defektsete P. putida tüvedega läbiviidud mutageneesi katsed näitasid järgnevat: 1.Alates teisest nädalast süsinikunälja tingimustes suureneb ühenukleotiidsete deletsioonide tekkesagedus olulisel määral. Ühenukleotiidsete deletsioonide teke P. putida rakkudes sõltub DNA polümeraas IV olemasolust. 2.P. putida rakkudes omavad aktinobakteritest pärineva geenikasseti poolt kodeeritud DNA polümeraasid DnaE2 ja ImuB mutageneesile vastupidist efekti. ImuB soodustab statsionaarse faasi populatsioonis nii asendusmutatsioonide kui ka ühenukleotiidsete deletsioonide teket samas kui DnaE2 olemasolu vastupidiselt kõigile senikirjeldatud DnaE2 homoloogidele, vähendab P. putida rakkudes asendusmutatsioonide tekkesagedust. 3.Tolueeni degradatsiooni võimaldava plasmiidi pWW0 poolt kodeeritud DNA polümeraas V homoloog suurendab statsionaarse faasi populatsioonis geneetiliselt enam kohastunud mutantide tekkesagedust

Involvement of Error-Prone DNA Polymerase IV in Stationary-Phase Mutagenesis in Pseudomonas putida

In this work we studied involvement of DNA polymerase IV (Pol IV) (encoded by the dinB gene) in stationary-phase mutagenesis in Pseudomonas putida. For this purpose we constructed a novel set of assay systems that allowed detection of different types of mutations (e.g., 1-bp deletions and different base substitutions) separately. A significant effect of Pol IV became apparent when the frequency of accumulation of 1-bp deletion mutations was compared in the P. putida wild-type strain and its Pol IV-defective dinB knockout derivative. Pol IV-dependent mutagenesis caused a remarkable increase (approximately 10-fold) in the frequency of accumulation of 1-bp deletion mutations on selective plates in wild-type P. putida populations starved for more than 1 week. No effect of Pol IV on the frequency of accumulation of base substitution mutations in starving P. putida cells was observed. The occurrence of 1-bp deletions in P. putida cells did not require a functional RecA protein. RecA independence of Pol IV-associated mutagenesis was also supported by data showing that transcription from the promoter of the P. putida dinB gene was not significantly influenced by the DNA damage-inducing agent mitomycin C. Therefore, we hypothesize that mechanisms different from the classical RecA-dependent SOS response could elevate Pol IV-dependent mutagenesis in starving P. putida cells

Oxidative DNA Damage Defense Systems in Avoidance of Stationary-Phase Mutagenesis in Pseudomonas putida▿

Oxidative damage of DNA is a source of mutation in living cells. Although all organisms have evolved mechanisms of defense against oxidative damage, little is known about these mechanisms in nonenteric bacteria, including pseudomonads. Here we have studied the involvement of oxidized guanine (GO) repair enzymes and DNA-protecting enzyme Dps in the avoidance of mutations in starving Pseudomonas putida. Additionally, we examined possible connections between the oxidative damage of DNA and involvement of the error-prone DNA polymerase (Pol)V homologue RulAB in stationary-phase mutagenesis in P. putida. Our results demonstrated that the GO repair enzymes MutY, MutM, and MutT are involved in the prevention of base substitution mutations in carbon-starved P. putida. Interestingly, the antimutator effect of MutT was dependent on the growth phase of bacteria. Although the lack of MutT caused a strong mutator phenotype under carbon starvation conditions for bacteria, only a twofold increased effect on the frequency of mutations was observed for growing bacteria. This indicates that MutT has a backup system which efficiently complements the absence of this enzyme in actively growing cells. The knockout of MutM affected only the spectrum of mutations but did not change mutation frequency. Dps is known to protect DNA from oxidative damage. We found that dps-defective P. putida cells were more sensitive to sudden exposure to hydrogen peroxide than wild-type cells. At the same time, the absence of Dps did not affect the accumulation of mutations in populations of starved bacteria. Thus, it is possible that the protective role of Dps becomes essential for genome integrity only when bacteria are exposed to exogenous agents that lead to oxidative DNA damage but not under physiological conditions. Introduction of the Y family DNA polymerase PolV homologue rulAB into P. putida increased the proportion of A-to-C and A-to-G base substitutions among mutations, which occurred under starvation conditions. Since PolV is known to perform translesion synthesis past damaged bases in DNA (e.g., some oxidized forms of adenine), our results may imply that adenine oxidation products are also an important source of mutation in starving bacteria

A DNA Polymerase V Homologue Encoded by TOL Plasmid pWW0 Confers Evolutionary Fitness on Pseudomonas putida under Conditions of Environmental Stress

Plasmids in conjunction with other mobile elements such as transposons are major players in the genetic adaptation of bacteria in response to changes in environment. Here we show that a large catabolic TOL plasmid, pWW0, from Pseudomonas putida carries genes (rulAB genes) encoding an error-prone DNA polymerase Pol V homologue which increase the survival of bacteria under conditions of accumulation of DNA damage. A study of population dynamics in stationary phase revealed that the presence of pWW0-derived rulAB genes in the bacterial genome allows the expression of a strong growth advantage in stationary phase (GASP) phenotype of P. putida. When rulAB-carrying cells from an 8-day-old culture were mixed with Pol V-negative cells from a 1-day-old culture, cells derived from the aged culture out-competed cells from the nonaged culture and overtook the whole culture. At the same time, bacteria from an aged culture lacking the rulAB genes were only partially able to out-compete cells from a fresh overnight culture of the parental P. putida strain. Thus, in addition to conferring resistance to DNA damage, the plasmid-encoded Pol V genes significantly increase the evolutionary fitness of bacteria during prolonged nutritional starvation of a P. putida population. The results of our study indicate that RecA is involved in the control of expression of the pWW0-encoded Pol V

Elevated Mutation Frequency in Surviving Populations of Carbon-Starved rpoS-Deficient Pseudomonas putida Is Caused by Reduced Expression of Superoxide Dismutase and Catalase▿

RpoS is a bacterial sigma factor of RNA polymerase which is involved in the expression of a large number of genes to facilitate survival under starvation conditions and other stresses. The results of our study demonstrate that the frequency of emergence of base substitution mutants is significantly increased in long-term-starved populations of rpoS-deficient Pseudomonas putida cells. The increasing effect of the lack of RpoS on the mutation frequency became apparent in both a plasmid-based test system measuring Phe+ reversion and a chromosomal rpoB system detecting rifampin-resistant mutants. The elevated mutation frequency coincided with the death of about 95% of the cells in a population of rpoS-deficient P. putida. Artificial overexpression of superoxide dismutase or catalase in the rpoS-deficient strain restored the survival of cells and resulted in a decline in the mutation frequency. This indicated that, compared to wild-type bacteria, rpoS-deficient cells are less protected against damage caused by reactive oxygen species. 7,8-Dihydro-8-oxoguanine (GO) is known to be one of the most stable and frequent base modifications caused by oxygen radical attack on DNA. However, the spectrum of base substitution mutations characterized in rpoS-deficient P. putida was different from that in bacteria lacking the GO repair system: it was broader and more similar to that identified in the wild-type strain. Interestingly, the formation of large deletions was also accompanied by a lack of RpoS. Thus, the accumulation of DNA damage other than GO elevates the frequency of mutation in these bacteria. It is known that oxidative damage of proteins and membrane components, but not that of DNA, is a major reason for the death of cells. Since the increased mutation frequency was associated with a decline in the viability of bacteria, we suppose that the elevation of the mutation frequency in the surviving population of carbon-starved rpoS-deficient P. putida may be caused both by oxidative damage of DNA and enzymes involved in DNA replication and repair fidelity