The reasons for inter-species differences in genome size is one of the major questions in biology, and has been under investigation by many researchers for many years. It is now recognized that this variation depends mainly on amplifications, deletions, and divergences of various repetitive sequences that are either distributed along the chromosomes or concentrated mainly in the heterochromatin (1–9). Relationships between genome size estimates and various physiological traits (rate of development, cell size, etc.) (10, 11) and environmental conditions (altitude, latitude, temperature, etc.) (12, 13) have suggested that natural selection could be involved in regulating genome size, although a change in size is primarily due to the genome's tolerance for repeated sequences (5) and its ability to mobilize them (14). Intra-species variation in genome size has also been shown to be associated with environmental conditions in plants, and seems to involve mobilization of some transposable elements in plants (15), Drosophila (14, 16), and pocket gophers (17). Any stressful conditions known to mobilize transposable elements, such as UV light, temperature, breeding conditions, etc. (18), are therefore factors potentially able to influence genome size. Walbot and Cullis (19, 20) thus propose that the mobilization of repeated DNA sequences may be a strategy for adaptating to a changing environment. It has even been reported that the amount of repeated elements in individual seeds depends on their position on the flowering head in Helianthus (21), although these differences seem to be very slight (6).
One important point in genome size measurement is that it is estimated by methods that are sensitive to chromatin structure, which modifies the accessibility of the DNA to the dye used. DNA contents estimated by flow cytometry, microspectrometry, or feulgen staining are usually correlated (6), and flow cytometry is increasingly viewed as an accurate method for estimating genome size if standard operating conditions are used (22). A correlation between estimates obtained using different methods is not in itself sufficient, however, to eliminate the possibility of an artifact, because DNA accessibility depends on complex interactions between intercalating or nonintercalating fluorochromes and DNA and various proteins. Variation in DNA accessibility due to the state of condensation of the chromatin has been reported in plants (21, 23), during spermatogenesis (24) and in frozen and thawed human spermatozoa (25), in mouse thymocyte nuclei (26), in ethanol-fixed cells (27), in dividing and stationary Euglena cells (28), during differentiation of Friend leukemia cells (29), and in DNAse I treated HeLa nuclei (30, 31). The exact impact of external environmental conditions on the genome size estimate therefore needs to be determined before a protocol can be defined that would permit reliable and comparable genome size estimates between individuals and populations within a given experiment and between different experiments. We investigated whether rearing conditions (temperature, humidity) and the conditions to which the extracted head nuclei were submitted before the DNA content was estimated were able to influence genome size estimate determined by flow cytometry in Drosophila melanogaster. We showed that the genome size estimate depended on the temperature and humidity of the larval rearing conditions, on the age of the adult flies, and on the temperature of the solution in which the fly nuclei were maintained before their introduction into the cytometer.
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Measurements of the genome size of species have often been determined from a single individual. It is becoming more and more evident, however, that genome size varies according to the population and even the individual concerned, and depends on various exogenous factors, the main effects of which seem to be the mobilization and increase in the copy number of transposable elements (2, 7, 32). Various correlations between genome size and latitude, altitude, temperature, light (9, 12, 13, 33), developmental rate (5), etc., are well documented, especially across species. Because the environment in which an organism grows seems to influence DNA content (6), it is concluded that genome size variation is an adaptation to changing environment. With the increasing use of flow cytometry based on intercalating fluorochromes, some of the correlations observed between genome size estimates and environmental factors may be artifacts, because DNA accessibility to the fluorochrome depends on chromatin structure, particularly its sensitivity to decondensation (23). Some slight fluctuations between individuals within populations and between populations may thus result, at least in part, from this kind of artifact, whereas differences between species are more likely really to involve natural selection and correspond to changes in genome size due to a change in the amount of DNA. We show here in Drosophila that the temperature and humidity level during development, the age of the adult flies measured, and the specific conditions of the buffers used for cytometric genome size estimation all directly influence the estimated genome size. We also checked the influence of the temperature at which the adult flies were maintained before measurement and the effects of ethyl ether anesthesia (data not shown). In both cases, some experiments showed statistically significant effects on genome size estimate, but the tendency was not reproducible, suggesting that various uncontrolled factors were interacting, sometimes in an antagonistic way, to influence the genome size estimate.
Drastic changes in the amount of transposable elements (TEs) are unlikely to be involved in such fluctuation in genome size, even though it is thought that organisms which develop at a slower rate tolerate more repeated DNA sequences (5, 34). This could be true for differences between species, but can hardly be applicable to immediate changes in genome size between individuals, depending on the temporary environmental conditions to which they are submitted. This does not mean, however, that transposable elements play no role in accounting for the differences in genome size between individuals and between populations. The TE content clearly differs between populations and species as a result of selection, and so may be related in some way to environmental conditions (14, 17). It is hard to imagine that a rapid change in TE content could be produced simply by changing the temperature at which the development of the flies takes place, the humidity of the medium, or the age of the flies, although such a possibility cannot be entirely ruled out by our experiments, and needs to be tested. A 14-fold increase in Tc1 somatic excision has indeed been reported during the lifespan of the nematode, Cænorhabditis elegans (35). It should be noted, however, that when our flies had food containing high levels of humidity, they had a longer developmental time (24 h more than under low humidity conditions), but their genome size estimate was smaller, which was contrary to what was expected. Similarly, there was no direct clear tendency towards a smaller or greater genome size estimate when development occurred at 17°C or 25°C. Moreover, the change in genome estimate depending on the temperature of the baths in which the nuclei were maintained before their DNA content was measured using the cytometer strongly suggests that what had changed was not the DNA content per se, but its estimation based on the intensity of fluorescence.
Fluctuations in genome size estimates of the kind observed in our experiments can thus simply be explained in terms of changes in the topology of chromatin, which modifies the accessibility of DNA to fluorochromes, especially to propidium iodide, which is often used in flow cytometry, as shown in various experiments (23, 26, 30, 31). Changes in DNA accessibility after a temperature treatment have been indeed reported for ethanol-fixed cells of rat thymocytes (27). The release of histones was blamed for the change in the binding of mithramycin as a result of a change in the three-dimensional structure of the chromatin (27). In Euglena cells, a change in fluorescence intensity was attributable to the exposure of chromatin-binding sites for ethidium bromide, which was related to the presence or absence of basic proteins (28). A decrease in DNA accessibility to different dyes, especially the DNA intercalators that unwind DNA, was observed during erythroid differentiation of leukemia cells. This effect disappeared after extraction of nuclear proteins using HCl (29). Histone H1 extraction increased the fluorescence intensity of isolated HeLa nuclei, because the binding sites had a higher affinity to the propidium iodide (30). Hence, the stainability of the DNA seems to depend greatly on nuclear decondensation (25) and the packaging with DNA-binding proteins, which also contributed to the difference in DNA stainability between spermatozoa from fertile and infertile men (36).
We must therefore be cautious when using flow cytometry to estimate DNA content, because many endogenous or exogenous factors, as well as the conditions to which the nuclei were submitted before being introduced into the cytometer (e.g., temperature, and pH of buffers ), may greatly influence the genome size estimate. Hence, before assigning a genome size value to a species, we need measurements for more than one population. In addition, the genome size estimate also depends on an appropriate standard of known genome size, which is a characteristic of every individual, in this case of the tetraodon fish, and of the environmental conditions to which the organisms to be analyzed were submitted. Strict controls and precautions are thus necessary before concluding that genome size is correlated with any specific environmental conditions.