Mutagenesis vol. 19 no. 5 © UK Environmental Mutagen Society 2004; all rights reserved.
DNA repair efficiency and thermotolerance in Drosophila melanogaster from ‘Evolution Canyon’
Institute of Evolution, University of Haifa, Mount Carmel, Haifa 31905, Israel
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The repair efficiency of four thermotolerant and four thermosensitive isofemale lines of Drosophila melanogaster originating from ‘Evolution Canyon’ (Mt Carmel, Israel) was tested using 2-acetylaminofluorene (2-AAF) as mutagen. First, males of the standard laboratory line Canton S were treated with either 2-AAF solution or control solution. Then, females of the ‘Evolution Canyon’ lines were crossed with treated (2-AAF or control solution) males and maintained at either 24 or 29°C. Arbitrary primed PCR fingerprinting was employed as a method for genomic damage analysis in the resulting progeny (by scoring the frequency of lost DNA bands in F1 progeny). Thermosensitive lines displayed significantly higher rates of change in the DNA fingerprint pattern after mutagenic presyngamic treatment followed by development at both temperatures, as well as after development under high temperature with no prior mutagenic treatment. The thermotolerant lines tended to show a lower level of mutation at both temperatures and after both treatments. One isofemale line showed a higher level of mutation at room temperature compared with increased temperature, after both control and mutagen treatment. The results suggest the existence of a relationship between DNA repair efficiency and thermotolerance, with thermotolerant lines tending to repair DNA more efficiently than thermosensitive ones.
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Introduction |
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In the course of evolution, organisms have had to adapt to diverse environmental conditions, including safeguarding living cells. Various mechanisms that protect the cell from protein denaturation might assist the organism in withstanding different stressful factors, like heat and drought (Chen,T. et al., 2002
; Lakhotia et al., 2002). DNA repair systems tend to minimize DNA damage caused by diverse external (environmental) and internal (stress-generated) mutagens (Friedberg et al., 1995). Genetic control and mechanisms of DNA repair have been among the main foci of molecular genetics during the last few decades and are rather well characterized in a few model organisms (Sancar and Tang, 1993; Friedberg et al., 1995; Vonarx et al., 1998). A few studies concerning the effect of environmental conditions on DNA repair efficiency have also been conducted, although the results seem to be controversial. Thus, while Raaphorst et al. (1999), Schmidt-Rose et al. (1999) and El-Awadi et al. (2001) showed decreased repair efficiency at high temperature, Kaszenman et al. (2000) reported that high temperature improves DNA repair efficiency. One may speculate that the genotype response to mutagenic treatment under stressful conditions may be affected by its stress resistance. Examining the interrelationships of stress resistance and DNA repair efficiency is a subject of growing interest among evolutionary biologists that may shed new light on the mechanisms of genetic adaptation to extreme environments (Korol et al., 1994; Krebs and Feder, 1998; Korol, 1999; Metzgar and Wills, 2000; Li et al., 2002; Rocha et al., 2002; Garbuz et al., 2003). In this study we attempted to test this kind of relationship using Drosophila melanogaster isofemale lines originating from ‘Evolution Canyon’ in Lower Nahal Oren, Mount Carmel, Israel.
The ‘European’ and ‘African’ north- and south-facing slopes (NFS and SFS) of ‘Evolution Canyon’ differ greatly in climatic regime, despite a very short interslope distance: 400 m at the top and 100 m at the bottom (Pavlicek et al., 2003). Although adult Drosophila can traverse several kilometers in a single day, populations on each slope have diverged in heat and desiccation tolerance, oviposition thermal preference, fluctuating asymmetry, mate preference and rates of mutation and recombination (Nevo et al., 1998; Korol et al., 2000; Iliadi et al., 2001; Michalak et al., 2001, and references therein). In the soil fungus Sordaria fimicola from ‘Evolution Canyon’, a higher recombination rate (Saleem et al., 2001) and a higher spontaneous mutation frequency (Lamb et al., 1998) were found in strains originating from the ‘African’ SFS compared with the ‘European’ NFS strains. The picture might be different where an environmental or chemical mutagen treatment is involved. Here the organism must deal with DNA damage caused by the mutagen that might activate different DNA repair pathways (the efficiency of repair enzymes might depend on temperature and, in turn, on the thermotolerance of the genotype). Thus, it was interesting to test whether or not a genotype-specific level of thermotolerance would modulate the efficiency of repair of DNA damage caused by a mutagen when the repair processes are conducted under normal and hot conditions.
To address the foregoing question, we applied the arbitrary primed polymerase chain reaction (APPCR) technique. APPCR is one of many methods that detect polymorphisms in fingerprints (Rojas et al., 1996). Recently, this method was applied to detecting DNA damage and repair. The initial phase of the experimental strategy involves treatment of adult males with mutagens, which so far have been 
-irradiation, ethylnitrosourea and 2-acetylaminofluorene (2-AAF) (Shimada and Shima, 1998; Lopez et al., 1999; Vasil'eva et al., 2001). The second phase includes crossing of the treated males with untreated females; the induced changes in the paternal chromosomes are supposed to be repaired (with different efficiency) in the fertilized eggs. The final stage is detecting the changes in the fingerprints of the F1 progeny by APPCR. This method is based on the knowledge that post-meiotic germ cells lack repair ability. The repair ability of the crossed females is detected in this case, based on scoring of the unrepaired damage that is displayed as changes in the fingerprints of the progeny. In this study we employed the APPCR method and 2-AAF as mutagen, known to cause frameshift (Broschard et al., 1999) and base substitution mutations (Vogel et al., 1996), to determine the relationship between heat resistance and DNA repair efficiency. The specific questions that we attempted to address in this study were as follows.
- Are there any differences in repair abilities of flies derived from the opposite slopes of ‘Evolution Canyon’?
- How do the post-treatment temperature conditions affect repair efficiency and how does the thermotolerance of a genotype modulate the joint effect of mutagen and increased temperature, if at all?
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Materials and methods |
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Drosophila melanogaster stocks
Drosophila melanogaster isofemale lines were constructed as follows. Drosophila melanogaster flies were collected during September 2000 from the two mid slopes of ‘Evolution Canyon’ 90 m above sea level (Station 2 on SFS and 6 on NFS). Each female was then placed in a separate vial containing Formula 4-24 instant Drosophila medium (Carolina Biological Supply Co.) and maintained at 24°C on a 12 h/12 h light/dark cycle. The 24 generations progeny of each original female constituted the isofemale lines employed for the analysis.
Heat tolerance test
Based on prior information about significant interslope differences in the ‘acquired’ thermotolerance (Rashkovetzky et al., in preparation), we decided to employ this test, rather than testing the reaction to ‘direct’ extreme heat shock, to characterize the material used in this study. To score the ‘acquired’ thermotolerance, a relatively mild heat pretreatment was followed by a more severe heat shock. Five-day-old flies from 14 isofemale lines originating from the opposite slopes of ‘Evolution Canyon’ were classified by sex under light CO2 anesthesia and placed in vials containing instant medium, 20 flies (females separated from males) in each vial. The vials containing flies were placed on a stand and incubated at 36°C in a water bath for 1 h. Then, after an additional 1 h at 24°C, the material was incubated for 1 h at 39°C and left at 24°C for 24 h. The number of live and dead flies was scored for each vial and pooled for each isofemale line of each gender. To test for significance of the effect of slope on thermotolerance we applied a log-linear analysis using the Statistica software (Statsoft, 1996).
Mutagenic and temperature treatment procedures
Males of the standard laboratory Drosophila melanogaster line Canton S, 3–4 days old, were starved for 4 h and then transferred to vials containing Whatman filter paper pieces (catalog no. 1001 150) saturated with either 4 mM 2-AAF (CAS no. 53-96-3; Sigma) dissolved in an aqueous solution containing 3% ethanol, 1% Tween 80 (CAS no. 9005-65-6; Sigma) and 5% sucrose or control solution containing all the above components except 2-AAF and kept in these vials for 24 h at 24°C. Treated males were crossed with virgin females from eight isofemale lines originating from the opposite slopes of ‘Evolution Canyon’ at male:female ratio of 1:2 and kept either at 24 or 29°C. The flies were allowed to lay eggs for 72 h, while every 24 h the parental flies were transferred to a new medium and eggs containing the old medium were maintained at 24°C until they hatched. As an additional control, untreated Canton S males receiving neither mutagen nor control solution were crossed with virgin females from the isofemale lines tested. Males from F1 progeny were collected for APPCR detection, with 13–48 flies from each of the above treatments. The experimental procedure is outlined in Figure 1.
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APPCR analysis
For each cross of an isofemale line with a treated Canton S male, a total of 80–208 F1 males were collected. Individual DNA extracts were prepared using the standard proteinase K protocol according to Gloor et al. (1993)
. The APPCR primer WB (5'-GTTAGGGAGCCGATAAAGAG) (Lopez et al., 1999) was labeled with a carbocyanine dye (Cy-5) on the 5'-end. The amplification conditions followed the conditions described by Lopez et al. (1999), with modifications for visualization using an ALFexpres II DNA automated sequencer. Each 25 µl reaction mixture contained 5 ng of template DNA, 1.2 µM WB primer, 2.5 µl of 10x PCR buffer containing 15 mM MgCl2, 124 µM each dNTP and 1 U Taq polymerase (Sigma). The reaction was initiated for 3 min of 94°C, followed by eight cycles of low stringency PCR (30 s at 94°C, 30 s at 40°C and 90 s at 72°C), 35 cycles of high stringency PCR (15 s at 94°C, 15 s at 55°C and 60 s at 72°C) and 10 min elongation at 72°C. PCR products were visualized by electrophoresis on denaturing sequencing gels with an ALFexpres II DNA automated sequencer using the Amersham Pharmacia Biotech protocol. The external standard used was ALFexpressTM SizerTM 50–500 bp (CAS no. 27-4539-01; Amersham Biosciences). Fragment sizes were determined with Alfwin Fragment Analyser 1.03 software (Amersham Pharmacia Biotech).
Statistical analysis
The analysis of bands was performed for each isofemale line separately, searching for any changes in the fingerprints such as lost bands, new bands or changes in band intensity and mobility shifts. A preliminary review of the APPCR amplimers revealed no changes in mobility or band intensity; likewise no new bands were detected. Hence, we decided to focus on lost bands. First, 25–27 reproducible monomorphic bands, 70–860 bp in size, were selected for each isofemale line after visualization in 36 Canton S males and in 16 F1 males from crosses between the tested isofemale lines and untreated Canton S males. We assumed that the genome affected by the mutagen was the genome of the Canton S males. Then, for the selected group of 25–27 bands, each F1 progeny was characterized for the number of bands that were lost due to treatment (damaged fraction) versus bands that were present (undamaged fraction). The data were analyzed by a log-linear model using Statistica software (Statsoft, 1996).
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Results |
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Heat tolerance
Eight isofemale lines from the ‘African’ SFS and six from the ‘European’ NFS were scored for heat resistance. The results of survival analysis after pretreatment at 36°C followed by treatment at 39°C (see Materials and methods) are presented in Table I. As can be seen from Table I, most of the SFS lines displayed a higher thermotolerance compared with the NFS lines. Usually, females tended to display a higher thermotolerance than males. This difference between sexes was observed especially among the more thermotolerant strains.
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The effects of factors ‘slope’ and ‘line’ were tested by a log-linear analysis using Statistica software. As can be seen from Table II, there is a highly significant interslope difference in the acquired thermotolerance (higher on the SFS). The results also point to significant within-slope variability, higher among SFS-derived lines (
2 = 154.7 for SFS and 24.3 for NFS, Fisher F-ratio F7,5 = 154.7/24.3 = 6.37, P < 0.029). Our intention was to take four tolerant and four susceptible lines in order to compare their reaction to mutagenic treatment combined with either elevated or normal maintenance temperature during after treatment development. The foregoing test revealed three lines from the SFS (S-13, S-3 and S-5) that appeared to be similar in their survival scores to NFS lines (i.e. displayed lowered thermotolerance compared with other SFS lines). Likewise, one line from the NFS (N-61) appeared to be similar to the SFS group. A posteriori reclassification by including S-13, S-3 and S-5 in the putatively ‘sensitive’ group and N-61 in the putatively ‘tolerant’ group gave a highly significant between-group difference in survivorship (2 = 103.15, d.f. = 1, P < 10–6) and lower intragroup diversity (2 = 14.0, d.f. = 5, P = 0.015 for thermotolerant lines; 2 = 48.7, d.f. = 7, P < 10–6 for susceptible lines) (see Table III). Therefore, for further experiments on DNA repair of mutagen-induced alterations using APPCR, eight isofemale lines were selected: four sensitive (N-65, N-612, N-62 and S-3) and four tolerant (S-15, S-2, S-9 and S-10). These lines were subjected to the four treatments: mutagenic and control solution, with maintenance of the material at 24 or 29°C until eclosion. The results are summarized in Table IV.
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Testing separate effects of mutagen treatment and hightemperature on the APPCR fingerprint
In order to address the main question of this study, whether there is any relationship between thermotolerance and DNA repair capability at normal and elevated temperature, the selected lines were subjected to four treatments: mutagen and control solutions, with subsequent maintenance of the material at 24 or 29°C until eclosion (Table IV). However, we first had to check whether or not the mutagenic treatment causes toxic effects that may bias our results. We estimated the toxicity by testing the effect of the mutagen on the number of offspring and sex ratio in two strains, S-9 and N-62. Canton S males were treated with 2-AAF or control solution as described in Materials and methods and crossed with females of strains S-9 and N-62. Each pair was left to lay eggs in a vial containing instant medium for 24 h. Then the flies were transferred to another vial and allowed to lay eggs for an additional 24 h. Afterwards, the males were removed and the females were allowed to lay eggs for another 24 h in a new vial. The female and male offspring were counted separately and the total number of offspring and the sex ratio were recorded. No significant effect of the mutagen on the number of offspring or the sex ratio was observed: the numbers of offspring per family in strain N-62 after mutagen and control treatment were 97 ± 14.6 and 76 ± 16.4, respectively, with a male:female ratio of 46.6:53.4 after mutagen treatment and 46.9:53.1 after control treatment. Strain S-9 gave 66 ± 18.8 offspring per family and a male:female ratio of 44.8:55.2 after mutagen treatment and 62 ± 13.9 offspring per family and a male:female ratio of 50.1:49.9 after control treatment.
Before analyzing the joint effect of the mutagen and temperature, we checked single factor effects: of mutagen treatment in the material maintained at 24°C and of increased temperature when a control solution (3% ethanol, 1% Tween 80 and 5% sucrose) was applied (Table IV). As can be seen, in both single factor treatments (either mutagen or temperature) a highly significant effect of thermotolerance was observed, with tolerant genotypes displaying a much lower level of lost bands in the DNA fingerprint compared with thermosensitive lines (Table IV and Figures 2 and 3). Mutagen treatment (factor M in Table V) was also highly effective, while no differences were revealed between thermotolerant and sensitive lines in reaction to mutagen in the material maintained at 24°C (2 = 28.19, d.f. = 1, P < 10–6 for factor M; 2 = 1.01, d.f. = 1, for interaction M x R). In contrast, temperature had no overall effect when applied without mutagen treatment, but this was due to the opposite directions of the temperature effects in tolerant and sensitive lines (for T x R interaction 2 = 11.99, d.f. = 1, P < 0.001; see Figure 3).
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The significant effect of the interaction T x R without mutagen treatment called for a further test: whether or not treatment with the control solution (3% ethanol, 1% Tween 80 and 5% sucrose) could cause changes in the DNA fingerprint. This test was conducted by comparison of DNA alterations in the progeny obtained from crosses of control-treated and untreated Canton S males with different isofemale lines (with the F1 progeny being maintained at room temperature throughout until eclosion). While the number of DNA alterations in the untreated Canton S cross was always 0, in the progeny of control-treated males crossed to isofemale lines S-3, N-65, S-10 and S-9 significant DNA alterations were observed (P = 0.005, P < 10–4, P = 0.005 and P = 0.05, respectively).
Combined effect of mutagen treatment, post-treatment temperature, genotype and thermotolerance on DNA alterations
The results of the multifactorial experiments are presented in Tables IV, VI and VII and in Figure 3. The significance of the separate effects of temperature, mutagen treatment, heat resistance and their interactions was analyzed using a log-linear model (Table VI). Only a slight overall effect of temperature was detected (2 = 4.58, P = 0.032), but the effects of thermotolerance, mutagen treatment and the interaction temperature x thermotolerance were found to be highly significant. Some indication of the significance of the interaction mutagen x thermotolerance x temperature was also obtained (2 = 5.07, P = 0.024). To analyze the effect of line and its interaction with other factors we had to deal separately with sensitive and tolerant lines (no nested analysis is available for the log-linear model). Analyzing the effect of line on the damaged fraction in thermosensitive and thermotolerant lines (Table VII) revealed significant variation among lines, with sensitive lines displaying far higher diversity than tolerant lines (2 = 311.8 and 35.6, for sensitive and tolerant lines, respectively, P < 10–6 in both cases). It is noteworthy that separate analysis of the two groups revealed a significant interaction mutagen treatment x temperature (2 = 5.91, P = 0.0151) for the reactions of tolerant but not sensitive lines, namely a much lower level of DNA fingerprint changes was observed after control solution treatment when the material was maintained at 29 compared with 24°C, whereas the differences between mutagenized material maintained at 24°C and that maintained at 29°C were trivial.
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While trying to figure out the source of the high variation among thermosensitive genotypes, we noted that these genotypes display a clear bimodal distribution. With respect to the level of changes in the DNA fingerprints, two lines (N-62 and N-612) were fairly close to the thermotolerant lines, whereas two other lines (N-65 and S-3) showed a several-fold higher output of changes (see Table IV). For this reason, we repeated the log analysis for thermosensitive lines complementing the factors ‘treatment’ and ‘temperature’ with a new one referred to as ‘repair capability’. This analysis revealed a significant effect of temperature, treatment and the interactions capability x temperature and capability x treatment. Hence, the ‘capable’ thermosensitive isofemale lines, although not affected by treatment, displayed a higher mutability at high temperature. And vice versa, ‘incapable’ lines were very much affected by treatment, but not temperature (Table VIII and Figure 3).
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Discussion |
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In this study we have used the APPCR technique to quantify the degree of genomic damage inherent in eight different isofemale lines of D.melanogaster originating from opposite slopes of ‘Evolution Canyon’. The genomic damage could be caused by the mutagen 2-AAF, by heat treatment or by both. The control solution that functioned as the mutagen solvent could also cause genomic damage to some degree. Spontaneous mutations might also occur due to the I-R system of hybrid dysgenesis. In this system D.melanogaster strains fall into two categories, denoted inducer (I) and reactive (R). Inducer strains carry the I-factor, which is a LINE-like retrotransposon. In the F1 females of a dysgenic cross between reactive females and inducer males transposition of active I elements may occur at a high rate, leading to high levels of mutations (Azou and Bregliano, 2001
). Whereas mutations resulting from the I-R system occur in F1 females, our testing scheme allows detection of genomic damage in males. To eliminate the effect of spontaneous mutations occurring for other reasons related to the cross between Canton S and our isofemale strains, we chose for analysis bands that appeared in F1 hybrid males from crosses between Canton S males and isofemale lines from ‘Evolution Canyon’ (see Materials and methods). The degree of genomic damage in the progeny reflects the DNA repair efficiency of the progenitors of the isofemale lines. Hence, we tried to assess the effect of heat resistance on DNA repair efficiency at normal and elevated temperatures.
2-AAF belongs to the class of aromatic amines. Its N-hydroxy derivative may become a highly reactive alkylating agent that reacts with guanine residues in DNA at the C-8 and N2 positions to yield N-(deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene and other derivatives (Friedberg et al., 1995). The repair machinery for such a lesion is nucleotide excision repair (NER). However, there are other mechanisms used by the cell that bypass this damage: trans-lesion synthesis, by which the replication machinery reads through the lesion, with an associated risk of fixing a mutation, such as frameshift mutations and base substitutions (Broschard et al., 1999; Tan et al., 2002); damage avoidance, a strategy that utilizes the information on the non-modified sister chromatid, thereby avoiding the mutagenic risk (Broschard et al., 1999, and references therein). Unrepaired DNA damage inherited by the progeny may reflect the efficiency of the NER machinery in the isofemale lines tested. Each of the other aforementioned machineries may cause changes in the information, expressed either as changes in the primer sequences if trans-lesion synthesis is involved or replacement of a certain primer site originating from the father by the homologous sequence originating from the mother if damage avoidance is involved. Both lead to the loss of bands.
NER is also the dark repair machinery of UV damages such as cyclobutane pyrimidine dimers and 6–4 pyrimidine pyrimidinone photoproducts. UV damages can be repaired either via photolysis, by the enzyme photolyase that requires light for its activity, or via NER process, which does not require light and hence it is called the ‘dark repair’ process. The slopes of ‘Evolution Canyon’ display dramatic physical and biotic contrasts at the micro scale (Nevo, 1997, 2001). The exposure of D.melanogaster to higher temperatures and levels of solar radiation and illumination on the SFS (Pavlicek et al., 2003) might confer on the ‘African’ SFS flies a higher heat and UV resistance than the ‘European’ NFS flies. On the other hand, the lush Euro-Asian vegetation of the NFS as compared with the Afro-Asian xeric savannah of the SFS might reduce the light significantly and lead to the predominance of a dark repair pathway of UV damage. Different causes might affect each individual or derived isofemale line differently and lead to the high variation in repair ability observed among the isofemale lines tested.
Heat tolerance
The heat resistance test revealed that five of eight SFS isofemale lines were scored as heat-resistant while five of six NFS isofemale lines were scored as heat-sensitive. These results corroborate the results of Nevo et al. (1998), in which flies from opposite slopes of ‘Evolution Canyon’ showed differences in oviposition temperature preferences, viability and longevity changes caused by short-term and lifetime temperature treatments and resistance to drought stress at different temperatures, and of Garbuz et al. (2003), that showed differences in thermotolerance among different Drosophila spp. inhabiting different climatic areas. However, three SFS isofemale lines were scored as heat-sensitive and one of them, S3, was selected for further DNA repair analysis.
DNA alterations after ‘control treatment’
In some isofemale lines DNA alterations were caused by control treatment at room temperature. As indicated above, the control solution was composed of ethanol (3%), Tween 80 (1%) and sucrose (5%). Among the control solution components, the only one known to cause DNA alterations is ethanol. It was found that chronic ethanol consumption by rats leads to DNA oxidative damage, which is not accompanied by a significant repair response (Karim et al., 2003). The oxidative damage, which is not accompanied by a repair response, might appear in genotypes S-9, N-65 and S-10 in which the control treatment at elevated temperature led to a lower mutation rate than at room temperature (24°C).
The combination of control treatment (ethanol) with high temperature might lead to a damage level sufficient to induce a repair response and might agree with the results of Rothkamm and Lobrich (2003), who revealed that double-strand breaks induced in cultures of non-dividing primary human fibroblasts by very low radiation doses (
1 mGy) remain unrepaired for many days, in strong contrast to the efficient double-strand breaks repair observed at higher doses. Ethanol was also found to cause DNA single- and double-strand breaks in Saccharomyces cerevisiae (Ristow et al., 1995). In most of the cases it might appear that the control treatment accompanied by an elevated temperature increased mutation rates among the heat-sensitive isofemale lines tested and lowered or did not effect mutation rate among heat-resistant isofemale lines as compared with control treatment at room temperature. Heat stress in the presence of oxygen causes oxidative stress, results in DNA damage via reactions with •OH (Davidson and Schiestl, 2001a,b, and references therein). The specific glycosylase enzymes encoded by NTG1, NTG2 and OGG1 in yeast are known to repair heat-dependent mutations, including a variety of pyrimidine and purine base lesions. Thermosensitive mutants of the yeast S.cerevisiae deficient in CoQ also displayed a higher number of mutation events after treatment at 50°C than the wild-type yeast strain. Other mutants, NDE1 and NDE2, like the mutant deficient in CoQ displayed low mutability after heat treatment despite extreme heat sensitivity (Davidson and Schiestl, 2001b). These findings might support the existence of various degrees of mutability among various thermosensitive strains. The correlation between mutability at high temperature and thermotolerance/sensitivity is probably dependent on specific enzymes that are usually, but not always, suppressed in thermosensitive isofemale lines.
DNA alterations caused by mutagen treatment
Among the heat-sensitive lines, two patterns were observed. The first pattern was observed in the ‘capable’ isofemale lines N-612 and N-62, where there was no effect of mutagenic treatment but there was an effect of temperature on mutation rate. In the isofemale line N-612 the control treatment combined with heat caused even higher mutagenesis than heat combined with 2-AAF treatment. Perhaps the combination of 2-AAF, heat and ethanol caused significant damage over the damage tolerance threshold that forced the isofemale line to activate the repair enzymes. In general, it appears that the repair system of the ‘capable’ genotypes is efficient enough to deal with the damage caused by 2-AAF (subject to NER) either at high temperature or at room temperature, but not sufficient to deal with heat-generated damage, oxidized purines and pyrimidines, which are repaired by glycosylases or by base excision repair.
A second pattern was characteristic of the genotypes S-3 and N-65 (‘incapable’) that displayed relatively high mutability at both temperatures and might be explained by poor NER ability. Three of the heat-resistant isofemale lines were unaffected by treatment or temperature. The highest scored heat-resistant isofemale line S-9 showed a lower mutation rate at high temperature after both control treatment and mutagen treatment. Perhaps, as mentioned above, room temperature might be a suboptimal temperature for repair of DNA damage caused by either ethanol or 2-AAF.
The goal of our study was to test whether the genetically determined stress tolerance would provide a higher DNA repair capability under stress or normal conditions after mutagen treatment. DNA repair mechanisms, which in our case (due to 2-AAF, ethanol or heat effects) could include NER (Heflich and Neft, 1994), non-homologous end joining (Pastink et al., 2001) and homologous recombination (Britt, 1995; Friedberg et al., 1995), might be affected by temperature because high temperatures cause protein denaturation. The activity of repair enzymes as well as other enzymes might be decreased and aggregation of other denaturated proteins in the vicinity might block the repair enzymes from reaching the site of damage. Heat shock proteins may minimize the effect of high temperature on repair. Heat-tolerant organisms produce heat shock proteins more efficiently, hence, their repair system might be less affected. It was also found that some repair enzymes are essential in the reaction to heat stress in the yeast S.cerevisiae (Reagan et al., 1995; Sommers et al., 1995) and in the bacteria Lactococcus lactis (Duwat et al., 1995). A core environmental stress response in fission yeast that is common to many types of stress, including heat, methyl methane sulfonate, oxidative stress, etc., has also been reported (Chen,D. et al., 2003), as has the involvement of chaperonins in NER in Escherichia coli (Zou et al., 1998).
This overlap may indicate that under the joint effect of mutagen and ecological stress, DNA repair mechanisms tend to operate more efficiently in some heat-tolerant genotypes than in sensitive ones, probably due to a higher expression of enzymes involved in both DNA repair and heat resistance. The observed results fit our expectation, despite the displayed diversity among isofemale lines: the heat-sensitive lines seemed to be more affected by heat treatment than heat-resistant isofemale lines. However, when 2-AAF treatment was involved, it seems that in thermosensitive isofemale lines damage could be repaired by a certain line either efficiently or inefficiently at both temperatures, while in thermotolerant lines the repair efficiency was higher or not different at high temperatures. So far, many studies have been conducted on the effect of high temperature, given prior to or following mutagen treatment, on repair efficiency and/or resistance to the mutagen (Raaphorst et al., 1999; Schmidt-Rose et al., 1999; Kaszenman et al., 2000; El-Awady et al., 2001). However, very few attempts have been made to evaluate the effect of resistance to stress on repair efficiency. The work of Beck and Dynlacht (2001) focused on extractability of the XRCC5 polypeptide after heating in thermotolerant as compared with thermosensitive U-1 human cells. The extractability of XRCC5 was higher in thermotolerant cells than in thermosensitive cells. This is putatively related to both the activity of the enzyme after heat treatment and to the DNA repair capability of thermotolerant cells. Two works by Korol et al. (reviewed in Korol et al., 1994) on the relationship between recombination rate, temperature and temperature adaptation in Drosophila and tomato revealed that recombination induced by heat treatment was negatively correlated with the resistance of flies or tomato to high temperatures. Tikhomirova (1980) found a lower rate of X chromosome loss in a heat-resistant genotype of D.melanogaster than in the standard Canton S strain at a high repair temperature given after ionizing radiation. Here we present direct extensive data indicating that in the fruit fly D.melanogaster there might be a correlation between the tolerance of the genotype to heat stress and the efficiency of repair of damaged sites in DNA, especially at high temperature. It might be very interesting to conduct similar tests of DNA repair efficiency and thermotolerance employing other mutagens, i.e. ionizing radiation using material collected from diverse ecological conditions.
The currently available limited experimental evidence on the relationship between repair efficiency, recombination frequency and resistance of the genotype to stress can be interpreted in terms of negative feedback of regulation of genetic variation in higher organisms (Korol et al., 1994). Stressful environments may increase the mutation (Lamb et al., 1998) and recombination (Saleem et al., 2001) rates due to ‘genomic stress’, thereby increasing genetic diversity (Hoffmann and Parsons, 1991; Korol, 1999; Satish et al., 2001). The foregoing experimental evidence allows us to conclude that the effect of environment on mutability and recombination may play an important role in genetic adaptation of natural populations (Korol, 1999). In addition to deleterious mutations, stress-induced mutability may also broaden the spectrum of beneficial mutations (Figure 4). Finally, increased genetic adaptation to environmental stress may decrease mutability and advance the population gene pool toward a new steady-state of genetic variability. How common such a system of negative feedback control of variability is in nature has yet to be ascertained.
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Acknowledgments |
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This work was conducted as part of a study by A.L. for a PhD degree and was supported by the Authority of Advanced Studies of Haifa University, BSF grant 9800443 and ISF grant 601-03-17.3. We acknowledge with thanks the three reviewers for constructive criticism and useful suggestions.
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1 To whom correspondence should be addressed. Tel: +1 972 4 8240449; Fax: +1 972 4 8246554; Email: korol{at}esti.haifa.ac.i
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References |
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Azou,Y. and Bregliano,J.C. (2001) I-R system of hybrid dysgenesis in Drosophila melanogaster: analysis of the mitochondrial DNA in reactive strains exhibiting different potentials for I factor transposition. Heredity, 86, 110–116.[CrossRef][ISI][Medline]
Beck,B.D. and Dynlacht,J.R. (2001) Heat-induced aggregation of XRCC5 (Ku80) in non-tolerant and thermo-tolerant cells. Radiat. Res., 156, 767–774.[CrossRef][ISI][Medline]
Britt,A.B. (1995) Repair of DNA damage induced by ultraviolet radiation. Plant Physiol., 108, 891–896.[CrossRef][ISI][Medline]
Broschard,T.H., Koffel-Schwartz,N. and Fuchs,R.P.P. (1999) Sequence dependent modulation of frameshift mutagenesis of NarI derived mutation hotspot. J. Mol. Biol., 288, 191–199.[CrossRef][ISI][Medline]
Chen,D., Toone,W.M., Mata,J., Lyne,R., Burns,G., Kivinen,K., Brazma,A., Jones,M. and Bahler,J. (2003) Global transcriptional responses of fission yeast to environmental stress. Mol. Biol. Cell, 14, 214–229.
Chen,T., Sun,H., Lu,J., Zhao,Y., Tao,D., Li,X. and Huang,B. (2002) Histone acetylation is involved in hsp70 gene transcription regulation in Drosophila melanogaster. Arch. Biochem. Biophys., 408, 171–176.[CrossRef][ISI][Medline]
Davidson,J.F. and Shiestl,R.H. (2001a) Cytotoxic and genotoxic consequences of heat stress are dependent on the presence of oxygen in Sacharomyces cervisiae. J. Bacteriol., 185, 4580–4587.
Davidson,J.F. and Shiestl,R.H. (2001b) Mitochondrial respiratory electron carriers are involved in oxidative stress during heat stress in Saccharomyces cerevisiae. Mol. Cell. Biol., 21, 8483–8489.
Duwat,P., Sourice,S., Ehrlich,S.D. and Gruss,A. (1995) RecA gene involvement in oxidative and thermal stress in Lactococcus lactis. Dev. Biol. Stand., 85, 455–467.
El-Awady,R.A., Dikomey,E. and Dahm-Daphi,J. (2001) Heat effects on DNA repair after ionizing radiation: hyperthermia commonly increases the number of nonrepaired double-strand breaks and structural rearrangements. Nucleic Acids Res., 29, 1960–1966.
Friedberg,E.C., Walker,G.C. and Siede,W. (1995) DNA Repair and Mutagenesis. American Society for Microbiology, Washington, DC.
Garbuz,D., Evgenev,M.B., Feder,M.E. and Zatsepina,O.G. (2003) Evolution of thermotolerance and the heat-shock response: evidence from inter/intraspecific comparison and interspecific hybridization in the virilis species group of Drosophila. I. Thermal phenotype. J. Exp. Biol., 206, 2399–2408.
Gloor,G.B., Preston,C.R., Johnson-Schlitz,D.M., Nassif,N.A., Phillis,R.W., Benz,W.K., Robertson,H.M. and Engels,W.R. (1993) Type I repressors of P element mobility. Genetics, 135, 81–95.[Abstract]
Heflich,R.H. and Neft,R.E. (1994) Genetic toxicity of 2-acetylaminofluorene, 2-aminofluorene and some of their metabolites and model metabolites. Mutat. Res., 318, 73–174.[CrossRef][ISI][Medline]
Hoffman,A.A. and Parsons,P.A. (1991) Evolutionary Genetics and Environmental Stress. Oxford University Press, Oxford.
Iliadi,K., Iliadi,N., Rashkovetsky,E., Minkov,I., Nevo,E. and Korol,A. (2001) Sexual and reproductive behaviour of Drosophila melanogaster from microclimatically interslope differentiated population of ‘Evolution Canyon’ (Mount Carmel, Israel). Proc. R. Soc. Lond. B, 268, 2365–2374.[Medline]
Karim,M.R., Wanibuchi,H., Wei,M., Morimura,K., Salim,E.I. and Fukushima,S. (2003) Enhancing risk of ethanol on MeIQx-induced rat hepatocarcinogenesis is accompanied with increased levels of cellular proliferation and oxidative stress. Cancer Lett., 192, 37–47.[CrossRef][ISI][Medline]
Kaszenman,D.J., Candreva,E.C. and Nunes,D. (2000) Cellular and molecular effects of bleomycin are modulated by heat shock in Saccharomyces cerevisiae. Mutat. Res., 459, 29–41.[ISI][Medline]
Korol,A.B. (1999) Selection for adaptive traits as a factor of recombination evolution: evidence from natural and experimental populations (review). In Wasser,S.P. (ed.) Evolutionary Theory and Processes: Modern Perspectives. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 31–53.
Korol,A.B., Preygel,I.A. and Preygel,S.I. (1994) Recombination Variability and Evolution. Chapman and Hall, London.
Korol,A.B., Rashkovetsky,E., Iliadi,K., Michalak,P., Ronin,Y. and Nevo,E. (2000) Non-random mating in Drosophila melanogaster laboratory populations derived from closely adjacent ecologically contrasting slopes at the ‘Evolution Canyon’, Israel. Proc. Natl Acad. Sci. USA, 97, 12637–12642.
Krebs,R.A. and Feder,M.E. (1998) Hsp70 and larval thermotolerance in Drosophila melanogaster: how much is enough and when is more too much? J. Insect Physiol., 44, 1091–1101.[CrossRef][ISI][Medline]
Lakhotia,S.C., Srivastava,P. and Prasanth,K.V. (2002) Regulation of heat shock proteins, Hsp70 and Hsp64, in heat-shocked Malpighian tubules of Drosophila melanogaster larvae. Cell Stress Chaperones, 7, 347–356.[CrossRef][ISI][Medline]
Lamb,B.C., Saleem,M., Scott,W., Thapa,N. and Nevo,E. (1998) Inherited and environmentally induced differences in mutation frequencies between wild strains of Sordaria fimicola from ‘Evolution Canyon’. Genetics, 149, 87–99.
Li,Y.C., Korol,A.B., Fahima,T., Beiles,A. and Nevo,E. (2002) Microsatellites: genomic distribution putative functions and mutational mechanisms: a review. Mol. Ecol., 11, 2453–2465.[CrossRef][Medline]
Lopez,A., Xamena,N., Cabre,O., Creus,A., Marcos,R. and Velazquez,A. (1999) Analysis of genomic damage in the mutagen-sensitive mus-201 mutant of Drosophila melanogaster by arbitrarily primed PCR (AP-PCR) fingerprints. Mutat. Res., 435, 63–75.[ISI][Medline]
Metzgar,D. and Wills,C. (2000) Evidence for the adaptive evolution of mutation rates. Cell, 101, 581–584.[CrossRef][ISI][Medline]
Michalak,P., Minkov,I., Helin,A., Lerman,D.N., Bettencourt,B.R., Feder,M.E., Korol,A.B. and Nevo,E. (2001) Genetic evidence for adaptation-driven incipient speciation of Drosophila melanogaster along a microclimatic contrast in ‘Evolution Canyon’ Israel. Proc. Natl Acad. Sci. USA, 98, 13195–13200.
Nevo,E. (1997) Evolution in action across phylogeny caused by microclimatic stresses at ‘Evolution Canyon’. Theor. Popul. Biol., 52, 231–243.[CrossRef][ISI][Medline]
Nevo,E. (2001) Evolution of genome-phenome diversity under environmental stress. Proc. Natl Acad. Sci. USA, 98, 6233–6240.
Nevo,E., Rashkovetsky,E., Pavlicek,T. and Korol,A. (1998) A complex adaptive syndrome in Drosophila caused by microclimatic contrasts. Heredity, 80, 9–16.
Pastink,A., Eeken,J.C.J. and Lohman,P.H.M. (2001) Genetic integrity and the repair of double-strand DNA breaks. Mutat. Res., 480/481, 37–50.
Pavlicek,T., Sharon,D., Kravchenko,V., Saaroni,H. and Nevo,E. (2003) Microclimatic interslope differences underlying biodiversity contrasts in ‘Evolution Canyon’, Mt. Carmel, Israel. Isr. J. Earth Sci., 52, 1–9.
Raaphorst,G.P., Ng,C.E. and Yang,D.P. (1999) Thermal radiosensitization and repair inhibition in human melanoma cells: a comparison of survival and DNA double-strand breaks. Int. J. Hyperthermia, 15, 17–27.[CrossRef][ISI][Medline]
Rashkovetsky,E., Lupu,A., Iliadi,K., Feder,M., Nevo,E. and Korol,A. (2004) Adaptive differentiation for thermotolerance in Drosophila melanogaster at the microsite, Nahal Oren Canyon (Mt. Carmel, Israel). In preparation.
Reagan,M.S., Pittenger,C., Siede,W. and Friedberg,E.S. (1995) Characterization of a mutant strain of Saccharomyces cerevisiae with a deletion of the RAD27 gene a structural homologue of the RAD2 nucleotide excision repair gene. J. Bacteriol., 177, 364–371.
Ristow,H., Seyfarth,A. and Lochmann,E.R. (1995) Chromosomal damages by ethanol and acetaldehyde in Saccharomyces cerevisiae as studied by pulsed field gel electrophoresis. Mutat. Res., 326, 165–170.[ISI][Medline]
Rocha,E.P., Matic,I. and Taddei,F. (2002) Over-representation of repeats in stress response genes: a strategy of increase versatility under stressful conditions? Nucleic Acids Res., 30, 1886–1894.
Rojas,M.O., De-Castro,J., Marino,G. and Wasserman,M. (1996) Detection of genomic polymorphism in Plasmodium falciparum using an arbitrarily primed PCR assay. J. Eukaryot. Microbiol., 43, 323–326.[ISI][Medline]
Rothkamm,K. and Lobrich,M. (2003) Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses. Proc. Natl Acad. Sci. USA, 100, 5057–5062.
Saleem,M., Lamb,B.C. and Nevo,E. (2001) Inherited differences in crossing over and gene conversion frequencies between wild strains of Sordaria fimicola from ‘Evolution Canyon’. Genetics, 159, 1573–1593.
Sancar,A. and Tang,M.S. (1993) Nucleotide excision repair. Photochem. Photobiol., 57, 905–921.[ISI][Medline]
Satish,N., Krugman,T., Vinogradova,O.N., Nevo,E. and Kashi,Y. (2001) Genome evolution of the cyanobacterium Nostoc linckia under sharp microclimatic divergence at ‘Evolution Canyon’, Israel. Microb. Ecol., 42, 306–316.[CrossRef][ISI][Medline]
Schmidt-Rose,T., Pollet,D., Will,K., Beremann,J. and Wittern,K.P. (1999) Analysis of UV-B-induced DNA damage and its repair in heat-shocked skin cells. J. Photochem. Photobiol. B, 53, 144–152.[CrossRef][Medline]
Shimada,A. and Shima,A. (1998) Combination of genomic DNA fingerprinting into the medaka specific-locus test system for studying environmental germ-line mutagenesis. Mutat. Res., 399, 149–165.[ISI][Medline]
Sommers,C.H., Miller,E.J., Dujon,B., Prakash,S. and Prakash,L. (1995) Conditional lethality of null mutations in RTH1 that encodes the yeast counterpart of a mammalian 5'- to 3' exonuclease required for lagging strand DNA synthesis in reconstituted systems. J. Biol. Chem., 270, 4193–4196.
Statsoft (1996) Statistica for Windows. Statsoft Inc., Tulsa, OK.
Tikhomirova,M.M. (1980) Relationship between the body's adaptation to heat and the modifying effect of extreme temperature on the effect of radiation. II. An analysis of the potential damages in a line adapted to heat. Genetika, 16, 290–298.[ISI][Medline]
Tan,X., Suzuki,N., Grollman,A.P. and Shibutani,S. (2002) Mutagenic events in Esherichia coli and mammalian cells generated in response to acetylaminofluorene-derived DNA adducts positioned in the NarI restriction enzyme site. Biochemistry, 41, 14255–14262.[CrossRef][Medline]
Vasil'eva,G.V., Bezlepkin,V.G., Lomaeva,M.G., Sirota,N.P. and Gaziev,A.I. (2001) AP-PCR assay of DNA alterations in the progeny of male mice exposed to low-level -radiation. DNA Repair, 485, 133–141.
Vogel,E.W., Nivrad,M.J., Ballering,L.A.B. et al. (1996) DNA damage and repair in mutagenesis and carcinogenesis: implications of structure–activity relationships for cross-species extrapolation. Mutat. Res., 113, 177–218.
Vonarx,E.J., Mitchell,H.L., Karthikeyan,R., Chatterjee,I. and Kunz,B.A. (1998) DNA repair in higher plants. Mutat. Res., 400, 187–200.[ISI][Medline]
Zou,W., Crowley,D.J. and Van Houten,B. (1998) Involvement of molecular chaperonins in nucleotide excision repair, DnaK leads to increased thermal stability of UvrA catalytic UvrB loading enhanced repair and increased UV resistance. J. Biol. Chem., 273, 12887–12892.
Received on September 25, 2003; revised on July 8, 2004; accepted on July 12, 2004.
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