Gianfranco Giorgi

Temperature‐related divergence in experimental populations of Drosophila melanogaster. II. Correlation between fitness and body dimensions

From a laboratory stock of Drosophila melanogaster (Oregon), reared for more than 20 years at 18° C, a new population was derived and maintained at 28° C for 8 years. The chromosomal and cytoplasmic contribution to genetic divergence between the two populations was estimated. Six body traits and reproductive fitness were taken into account. The third chromosome is responsible for the adaptive difference for temperature between the two lines. Temperature‐selected genes which control body size are located on the second and third chromosomes, although the contribution of each chromosome depends on the environment in which the flies develop. The correlation between the chromosomal and cytoplasmic contributions to different traits and fitness, changes with temperature. At 28° C the correlation between fitness and each body trait is proportional to the response to selection exhibited by each of them, but this is not true at 18° C. Body size has, therefore, an adaptive significance in relation to temperature, which is expressed only in the environment where selection occurs. Cytoplasmic genes affect almost all characters to an extent similar to that of chromosomal genes. Inter‐chromosomal and nucleo‐cytoplasmic interactions are present and also change with temperature. In general, genes selected in a given environment produce greater phenotypic changes in that environment than in another. The population that experienced both temperatures is fitter in both environments, suggesting that the capacity to adapt to warm temperatures depends on genes other than those which are involved in the adaptation to cold.

Temperature-related divergence in experimental populations of Drosophila melanogaster. III. Fourier and centroid analysis of wing shape and relationship between shape variation and fitness.

From a laboratory stock of Drosophila melanogaster (Oregon), reared for more than 20 years at 18°C, two new populations were derived and maintained at 25° and 28°C for 8 years. The chromosomal and cytoplasmic contribution to genetic divergence between the two more extreme populations was estimated at 18°C and 28°C. Wing shape and two fitness components (fecundity and fertility) were taken into account. Fourier descriptors and the position of the centroid were taken as indicators either of wing shape variation, determined by a different response of the two wing compartments to temperature selection, or of wing shape variation determined by both compartments. The descriptors appear to be good characters: they show a variability which is genetically controlled and ascribable to genes located on specific chromosomes. The third chromosome is responsible for the adaptive difference to temperature. The genes which control wing shape are located on the second and third chromosome, although the contribution of each chromosome depends on the environment in which the flies develop. Cytoplasmic genes display an effect as large as that of chromosomes, and nucleus × cytoplasm interaction is present. The correlation between the genetic contributions to compartment‐dependent wing shape variation and the contributions to fitness is highly significant, especially at 28°C. Wing shape has, therefore, an adaptive significance in relation to temperature, which is particularly expressed in the environment where selection occurred.

DEVELOPMENTAL CONSTRAINTS AND WING SHAPE VARIATION IN NATURAL POPULATIONS OF DROSOPHILA MELANOGASTER

The body sizes and shapes of Poikilothermic animals generally show clinal variation with latitude. Among the environmental factors responsible for the cline, temperature seems to be the most probable candidate. In the present work we analysed natural populations of Drosophila melanogaster collected at different geographical localities to determine whether the same selective forces acting on wing development in the laboratory are also at work in the wild. We show that the temperature selection acting on wing development in the laboratory is only one of the selective forces operating in the wild. The size differences between natural populations seem to depend exclusively on cell number whereas they depend on cell area in the laboratory. The two wing compartments behave as distinct units of selection subjected to different genetic control, confirming our previous observations on laboratory populations. In addition, subunits of development defined as regions of cell proliferation centres restricted within longitudinal veins can, in turn, be considered as subunits of selection. Their interaction during development and continuous natural selection around an optimum could explain the high wing shape stability generally found in natural populations.

LINE-1 retrotransposition in human neuroblastoma cells is affected by oxidative stress

Long interspersed element-1s (LINE-1 or L1s) are abundant retrotransposons that occur in mammalian genomes and that can cause insertional mutagenesis and genomic instability. L1 activity is generally repressed in most cells and tissues but has been found in some embryonic cells and, in particular, in neural progenitors. Moreover, L1 retrotransposition can be induced by several DNA-damaging agents. We have carried out experiments to verify whether L1 retrotransposition is affected by oxidative DNA damage, which plays a role in a range of human diseases, including cancer and inflammatory and neurodegenerative disease. To this purpose, BE(2)C neuroblastoma cells, which are thought to represent embryonic precursors of sympathetic neurons, have been treated with hydrogen peroxide and subjected to an in vitro retrotransposition assay involving an episomal L1RP element tagged with enhanced green fluorescent protein. Our results indicate that hydrogen peroxide treatment induces an increase in the retrotransposition of transiently transfected L1RP and an increase in the expression of endogenous L1 transcripts. An increase of γ-H2AX foci and changes in the mRNA levels of MRE11, RAD50, NBN and ERCC1 (all involved in DNA repair) have also been found. Thus, oxidative stress can cause L1 dysregulation.

Developmental constraints in the Drosophila wing

Selection experiments for shortening the four longitudinal veins in a wild population of Drosophila melanogaster have been performed to evaluate how a local change is integrated in the wing development. Our results show that, though many units of selection seem to exist within a given organ, these are strongly constrained within the developmental programme, in such a way that only some predictable forms are expected. The results are discussed in terms of the ‘Entelechia’ model proposed by Garcia-Bellido in which the intercalarity of positional values promoted by ‘martial’ genes in a given organ is the driving force for controlled cell proliferation.

Correlation between characters as related to developmental pattern in Drosophila

In order to better understand the genetic basis of some body traits and their correlations in Drosophila, in relation to their developmental history, a biometrical study was performed on three lines selected for short wing (fourth vein) and a control strain.The correlated response to selection for short wing and four body traits (thorax length and width, scutellar length, head width) and of eight other dimensional wing traits was considered.The results show a strong correlated response to selection of all wing traits, low correlations for the thorax characters, while head width remains relatively constant. Two groups of wing characters, corresponding to compartments of development, show different levels of covariation with the selected trait, the covariation being greater when the characters included in the same compartment of the selected trait are considered.The results are discussed in terms of developmental genetics of Drosophila and suggest that quantitative studies may be suitable for studying the rôle of interactions between sets of genes controlling development.

Synergic effect of retinoic acid and extremely low frequency magnetic field exposure on human neuroblastoma cell line BE(2)C

The aim of the present study was to assess whether exposure to a sinusoidal extremely low frequency magnetic field (ELF-MF; 50 Hz, 1 mT) can affect proliferation and differentiation in the human neuroblastoma cell line BE(2)C, which is representative of high risk neuroblastomas. Cells were subjected to ELF-MF exposure in the presence or absence of a neuronal differentiating agent (all-trans-retinoic acid, ATRA) for 24-72 h. In each experiment, ELF-MF-exposed samples were compared to sham-exposed samples. Cells exposed to ELF-MF combined with retinoic treatment showed a decreased cellular proliferation and an increased proportion of G(0)/G(1) phase cells compared to cells exposed to either treatment alone. Moreover, ELF-MF- and ATRA-treated cells showed more differentiated morphological traits (a higher neurite number/cell, an increased neurite length), together with a significant increase of mRNA levels of p21(WAF1/CIP1) and cdk5 genes, both involved in neuronal differentiation. In addition, the expression of cyp19 gene, which is involved both in neuronal differentiation and stress response, was evaluated; cyp19 gene expression was enhanced by ATRA treatment and significantly enhanced further by ELF-MF exposure combined with ATRA. In conclusion, our data suggest that ELF-MF exposure can strengthen ATRA effects on neuroblastoma cells.

Effect of extremely low frequency magnetic field exposure on DNA transposition in relation to frequency, wave shape and exposure time

Purpose: To examine the effect of extremely low frequency magnetic field (ELF-MF) exposure on transposon (Tn) mobility in relation to the exposure time, the frequency and the wave shape of the field applied. Materials and methods: Two Escherichia coli model systems were used: (1) Cells unable to express β-galactosidase (LacZ-), containing a mini-transposon Tn10 element able to give ability to express β-galactosidase (LacZ+) upon its transposition; therefore in these cells transposition activity can be evaluated by analysing LacZ+ clones; (2) cells carrying Fertility plasmid (F+), and a Tn5 element located on the chromosome; therefore in these cells transposition activity can be estimated by a bacterial conjugation assay. Cells were exposed to sinusoidal (SiMF) or pulsed-square wave (PMF) magnetic fields of various frequencies (20, 50, 75 Hz) and for different exposure times (15 and 90 min). Results: Both mini-Tn10 and Tn5 transposition decreased under SiMF and increased under PMF, as compared to sham exposure control. No significant difference was found between frequencies and between exposure times. Conclusions: ELF-MF exposure affects transposition activity and the effects critically depend on the wave shape of the field, but not on the frequency and the exposure time, at least in the range observed.

Evaluation of LINE-1 mobility in neuroblastoma cells by in vitro retrotransposition reporter assay: FACS analysis can detect only the tip of the iceberg of the inserted L1 elements

Long Interspersed Nuclear Elements (L1) are retroelements generally repressed in most differentiated somatic cells. Their activity has been observed in some undifferentiated and tumour cells and could be involved in tumour onset and progression. Growing evidences show that the L1 activation can occur in neuronal precursor cells during differentiation process. Neuroblastoma is a tumour originating from neuronal precursor cells, and, although the molecular basis of its progression is still poorly understood, the implication of L1 activation has not yet been investigated. In this study L1 mobility in neuroblastoma BE(2)C cells was assessed using the in vitro retrotransposition assay consisting in an episomal EGFP-tagged L1(RP) element, whose mobility can be evaluated by cytofluorimetric analysis (FACS) of EGFP expression. FACS results have shown a low retrotransposition activity. To detect L1(RP) integrated in transcriptionally repressed genomic sites, both a cell treatment with a stimulator of reporter gene promoter, and a quantitative Real-Time PCR analysis were performed. A retrotransposition activity ten and one thousand times that of FACS was found, respectively. These results point out that the real rate of L1 retrotransposition events in tumour cells might be considerably higher than that reported so far by evaluating only the reporter gene expression.

Assessing LINE-1 retrotransposition activity in neuroblastoma cells exposed to extremely low-frequency pulsed magnetic fields.

Mobile genetic elements represent an important source of mutation and genomic instability, and their activity can be influenced by several chemical and physical agents. In this research we address the question whether exposure to extremely low-frequency pulsed magnetic fields (EMF-PMF) could affect the mobility of the human LINE-1(RP) retrotransposon. To this purpose, an in vitro retrotransposition assay was used on human neuroblastoma BE(2) cells exposed for 48h to 1mT, 50Hz PMF, or sham-exposed. Moreover, since it is well known that retrotransposition causes DNA double-strand breaks (DSB), an estimation of γ-H2AX foci, which is a marker of DNA DSB, was carried out on PMF- and sham-exposed samples. The results show that PMF-exposed cells had a lower number of both retrotransposition events and DNA DSB compared with sham-exposed samples. These results suggest that exposure to PMF can interfere with retrotransposition activity by inducing a decrease of retrotransposition events.