Behavior of the hobo Transposable Element with Regard to TPE Repeats in Transgenic Lines of Drosophila melanogaster

Sémi Souames*, Claude Bazin{dagger}, Eric Bonnivard* and Dominique Higuet*,

* Institut Jacques Monod, Universités Paris 6 et 7, CNRS, Laboratoire Dynamique du Génome et Evolution, Paris, France
{dagger} CNRS. Laboratoire Population, Génétique et Evolution, Gif sur Yvette, France

Correspondence: E-mail: higuet{at}ccr.jussieu.fr.


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
The hobo transposable element of Drosophila melanogaster is known to induce a hybrid dysgenesis syndrome. Moreover it displays a polymorphism of a microsatellite in its coding region: TPE repeats. In European populations, surveys of the distribution of hobo elements with regard to TPE repeats revealed that the 5TPE element is distributed along a frequency gradient, and it is even more frequent than the 3TPE element in Western populations. This suggests that the invasive ability of the hobo elements could be related to the number of TPE repeats they contain. To test this hypothesis we monitored the evolution of 16 lines derived from five initial independent transgenic lines bearing the 3TPE element and/or the 5TPE element. Four lines bearing 5TPE elements and four bearing 3TPE elements were used as a noncompetitive genetic background to compare the evolution of the 5TPE element to that of the 3TPE element. Eight lines bearing both elements provided a competitive genetic context to study potential interactions between these two elements. We studied genetic and molecular aspects of the first 20 generations. At the molecular level, we showed that the 5TPE element is able to spread within the genome at least as efficiently as the 3TPE element. Surprisingly, at the genetic level we found that the 5TPE element is less active than the 3TPE element, and moreover may be able to regulate the activity of the 3TPE element. Our findings suggest that the invasive potential of the 5TPE element could be due not only to its intrinsic transposition capacity but also to a regulatory potential.

Key Words: hybrid dysgenesis • hobo element • permissivity • microsatellite


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
The hobo transposable element of Drosophila melanogaster, which is known to induce a hybrid dysgenesis syndrome in the species, transposes via a DNA intermediate and is a member of the hobo-Ac-Tam family (hAT; Calvi, Hong, and Gelbart 1991). The ORF1 of the hobo element that encodes the putative transposase contains a polymorphic region (S region) consisting of a tandemly repeated "ACTCCAGAA" sequence. This sequence encodes a threonine-proline-glutamic acid motif (TPE motif). In the first hobo element to be described, hobo108, 10 perfect repeats were revealed, whereas HFL1, the autonomous reference element, contains three perfect TPE repeats. These perfect repeats are flanked by five degenerate ones, three in the 5' position and two in the 3' position (Streck, MacGaffey, and Beckendorf 1986; Calvi, Hong, and Gelbart 1991). Moreover, Bonnivard et al. (2000) and Bonnivard, Bazin, and Higuet (2002) detected hobo elements with two to nine TPE repeats in natural populations that represent a unique case of polymorphism among transposable elements. On the basis of the distribution of this polymorphism, they propose a two-step invasion scenario. In a first wave, the 3TPE element, which is distributed worldwide, completely invaded the natural populations. New hobo elements then appeared as a result of mutations of the TPE repeats. We have previously shown that in transgenic lines of D. melanogaster the high polymorphism of the S region is the consequence of a high mutation rate of the TPE repeats, estimated to be 4.5 x 10–4 per copy and per generation (Souames et al. 2003). Most of the new elements that appeared in natural populations were locally present at very low frequencies, apart from the 4TPE and 5TPE elements. Both these elements are present at high frequencies in natural populations, and they could have triggered a new stage of invasion. Thus, the invasive capacity of hobo elements could be related to the number of TPE repeats.

The behavior of hobo elements with regard to the TPE repeats remains to be elucidated. Indeed, the 3TPE hobo element (HFL1) has been shown experimentally to be active, which is consistent with its worldwide distribution. Ladevèze et al. (1994, 1998, 2001) have shown in transgenic lines that this element is able to spread within the genome of D. melanogaster. In other respects, in European populations the 5TPE element is distributed along a centrifugal frequency gradient. In Western European populations this element is even more frequent than the 3TPE element (Bonnivard, Bazin, and Higuet 2002), and the authors suggest that the 5TPE element could be replacing the 3TPE element in European populations. We have shown that the 5TPE element is active and has been transposed in transgenic lines of D. melanogaster (Souames et al. 2003). Then, the situation observed in natural populations could be the consequence of the intrinsic ability of the 5TPE hobo element to transpose.

In this article we report an investigation of the capacity of 5TPE hobo elements to invade transgenic lines of D. melanogaster. To detect any interactions, we carried out our investigation in two different genetic contexts, a noncompetitive genetic context, in which 5TPE or 3TPE elements evolved in isolation, and a competitive genetic context, in which they evolved together in the same line. To survey the evolution of hobo elements in the lines over time, molecular and functional parameters have been used. At the molecular level we surveyed the evolution over time of the mean copy number per fly of full-sized hobo elements. At the functional level, we used the ability of hobo elements to induce a hybrid dysgenesis syndrome. This occurs in the F1 progeny of dysgenic crosses involving females devoid of hobo elements (E females) and males bearing hobo elements (H males). Increasing mutation rate, chromosomal rearrangements and breakage, male recombination, and thermosensitive sterility at 23°–25°C can be observed (Blackman et al. 1987; Yannopoulos et al. 1987; Stamatis et al. 1989). Complete agonadism is responsible for the thermosensitive sterility symptom (GD sterility). From these symptoms, we used the GD sterility to estimate the activity and the permissivity in each line. The hobo activity is defined as the ability of hobo elements to induce GD sterility in the dysgenic cross. The permissivity is the ability of the females to allow hobo activity in their progeny when they are crossed with males harboring active hobo elements. A low level of GD sterility reflects a low level of permissivity that indicates a regulatory potential of the tested line. A high level of GD sterility reflects a high level of permissivity that signals the absence of a regulatory potential. We show that the ability of the 5TPE element to invade the genome of D. melanogaster could be related not only to its intrinsic transposition capacity but also to a putative regulatory effect on the 3TPE element.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Strains
The yw and zola strains are devoid of hobo elements (E strains). The yw strain was used as the control for molecular and permissivity analyses, because hobo elements have been introduced into that strain. The zola strain was used in dysgenic crosses because it is a highly permissive strain that allows a high level of expression of the gonadal dysgenesis (GD) symptom.

The CyHBL1 strain was used as the reference strain for estimating the mean copy number of full-sized hobo elements in the lines, because it contains one copy of the HFL1 element per diploid genome (Calvi and Gelbart 1994). The 23.5/Cy MRF reference strain was used as a source of hobo transposase in the permissivity analysis (Yannopoulos et al. 1983; 1987; Bazin and Higuet 1996). The 23.5/Cy MRF reference strain harbors 3TPE hobo elements.

Transgenic Lines: Constructs and Microinjections
The pHfl1 plasmid is a complete 3TPE hobo element cloned in the pBlueScript (Calvi, Hong, and Gelbart 1991). The pHfl5 is a 5TPE element obtained by substituting an S region with 5TPE repeats for the S region of pHfl1 (Béatrice Denis, personal communication). The pHfl plasmids (1 and 5) were digested by two enzymes, KpnI and NaeI, to obtain a restriction fragment that contains the complete hobo element plus 0.5-kb of genomic DNA and a small fragment of pBlueScript. These 3.4-kb fragments, containing either the 3TPE hobo element or the 5TPE hobo element, were inserted into the KpnI/StuI pUAST plasmid fragment. This plasmid, described in Brand and Perrimon (1993), contains the 5' and the 3' ends of the P element with the miniwhite reporter gene. The resulting constructs are pP{3TPE hobo, white+} and pP{5TPE hobo, white+}.

Dechorionated early embryos from the ME yw strain (devoid of P and hobo elements) were microinjected with pP{3TPE hobo, white+} and pP{5TPE hobo, white+} constructs using the {Delta}2.3 helper plasmid as a P transposase source (Laski, Rio, and Rubin 1986). The F0 survival adults were crossed with individuals from the yw strain on standard medium at 25°C. The individual F1 progeny were screened for eye color that ranged from pale yellow to orange. Thus, brothers and sisters with a given eye color were crossed to establish a transgenic line. Four independent transgenic lines with the 3TPE element (lines {alpha}, ß, {chi}, and {delta}) and one line with the 5TPE element (line {kappa}) were obtained. It was possible to distinguish heterozygous and homozygous individuals using an eye color screening.

hobo T-lines
One hundred hobo T-lines were established in the laboratory from the four independent transgenic lines bearing the 3TPE element and the transgenic line bearing the 5TPE element. These lines were maintained in vials at 25°C and at each generation parents (50 to 100 individuals) were discarded after 24 hours of egg-laying. Thirty hobo 3T-lines were obtained from a single pair of heterozygous flies of the initial transgenic lines bearing the 3TPE hobo element, among which four were chosen to be analyzed. The 3-A and 3-B lines were obtained from the same progenitor transgenic line, whereas 3-C and 3-D lines were independently obtained from two other transgenic lines (table 1). Thirty hobo 5T-lines were obtained from a single pair of heterozygous flies of the initial transgenic line bearing the 5TPE hobo element. We chose four lines (5-A to 5-D) to be analyzed (table 1). These hobo 3T-lines and hobo 5T-lines provide a noncompetitive genetic context in which to study hobo behavior.


View this table:
[in this window]
[in a new window]
 
Table 1 Consitution of the hobo T-lines.

 
To study potential interactions between the 3TPE and the 5TPE elements, 20 hobo 35T-lines and 20 hobo 53T-lines were obtained by crossing a single heterozygous individual from the initial transgenic lines that bore the 3TPE hobo element with a single individual from the initial transgenic line that bore the 5TPE element. In hobo 35T-lines the 3TPE element originated on the maternal side, whereas in hobo 53T-lines it was the 5TPE element that originated on the maternal side. Four hobo 35T-lines (35-A to 35-D) and four hobo 53T-lines (53-A to 53-D) were chosen to provide a competitive genetic context. In these hobo 35T-lines and 53T-lines, the 3TPE element of each of the four lines originated from an independent transgenic line containing the 3TPE element (table 1). These 16 T-lines lines were kept in mass cultures at 25°C, and at each generation parents (200 to 300 individuals in bottles) were discarded after 24 hours of egg-laying.

Functional Analysis of the Behavior of hobo Elements
One of the parameters of the hybrid dysgenesis syndrome induced by hobo elements that we used to estimate the activity of hobo elements and the permissivity of the lines was GD sterility, measured by atrophied gonads (i.e., complete agonadism as defined in the P-M system by Kidwell and Novy 1979). In other respects, GD sterility has also been used to estimate the intra-strain GD sterility that reflects the result of both hobo activity and permissivity.

Intra-strain GD Sterility Assay
The intra-strain GD sterility (intraGD) was measured at the 11th, 17th, and 20th generations after the establishment of the lines. A mean number of 50 females (at least 35 females) per line and per generation were sampled and fed for 2 days on standard medium at 25°C before being dissected. The intraGD was estimated as the percentage of dystrophic ovaries: (number dystrophic ovaries/number dissected) x 100.

Activity of the hobo T-lines
In each hobo T-line, the hobo activity (induced GD sterility) was measured in the F1 progeny of crosses between five zola strain females (E females) and five hobo T-line males (males under test). Crosses were performed at 25°C for six generations (5th, 7th, 11th, 14th, 17th, and 20th). At each generation, each line was studied through two replicates, and a mean of 50 females per replicate were dissected.

Permissivity of the hobo T-lines
The permissivity of each hobo T-line was measured in the F1 female progeny of the crosses between females from the T-lines (females under test) and males from the 23.5/Cy MRF reference strain (H males) and based on the GD sterility induced by the paternal 23.5 MRF chromosome. The analyses were performed twice after the 11th generation. Eight of the 16 mass-cultured T-lines (5-B, 5-C, 3-C, 3-D, 53-B, 53-D, 35-A, and 35-D) and four other hobo T-lines maintained in vials (5–10, 3–28, 53–10, and 35–4; table 1) were analyzed. The GD sterility was measured by determining the number of dystrophic ovaries. The control cross of permissivity involved females from the yw strain used for the transformation experiment.

Molecular Analysis of the Behavior of hobo Elements
Southern Blot Analysis
DNA extractions were performed on 30 females from each hobo T-line according to the Junakovic, Caneva, and Ballario (1984) protocol. Two micrograms of DNA were digested with the XhoI enzyme that generates a 2.6-kb internal fragment corresponding to the complete hobo element. Six generations (5th, 7th, 11th, 14th, 17th, and 20th) were tested. The standard Southern blot technique (Sambrook, Fritsch, and Maniatis 1989) was used to estimate the mean copy number of full-sized hobo elements in the lines. To assess the amount of DNA present, the membranes were hybridized with a white gene probe that reveals the endogenous white gene. The relative amount of DNA in the lanes was estimated by scanning densitometry, using the signal intensities of the endogenous white gene referring to the yw strain. To estimate the number of full-sized hobo elements, the membranes were then hybridized with a hobo probe, an eluted XhoI internal fragment from the hobo108 plasmid (Streck, MacGaffey, and Beckendorf 1986). The mean copy number of full-sized elements per fly in a line corresponds to the ratio of the hobo108 signal intensities of the hobo T-line and the CyHBL1 strain, corrected by the ratio of DNA amount.

Polymerase Chain Reaction Analysis
The lines were established from heterozygous individuals, and two main classes were expected: [H] class individuals containing hobo elements and [E] class individuals devoid of these elements. Actually, the [H] class consists of one class in the hobo 3T-lines and one in the hobo 5T-lines, the [3] and [5] classes, respectively, whereas it consisted of [3], [5], and [3,5] classes in hobo 35T-lines and hobo 53T-lines. To estimate the proportions of the different classes in the lines, samples of 10 flies were individually analyzed at the 7th and the 20th generations by h11-h6 polymerase chain reaction (PCR) amplification (Bazin and Higuet 1996). The h11 (1756–1774) and h6 (2148–2168) primers are specific primers of the S region that contains the TPE repeats. DNA extractions were performed following the DiFranco et al. protocol (1995), and PCR products were screened on a 2% agarose gel allowed to migrate on ice for 3 hours at 100 V.

Statistical Analysis
Hierarchical analyses of variance (ANOVA), followed by Duncan multiple pairwise comparisons, were performed on the activity data (after arcsin transformation), and on the mean copy number of full-sized hobo elements. We performed the r coefficient of correlation test on arcsin transformed data of the activity and the mean copy number of full-sized elements. To compare the proportions of classes of individuals in the lines, we performed exact Fisher tests with the level of significance corrected using the sequential Bonferroni technique (Rice 1989). To compare the levels of permissivity, we performed a Chi-square test. All statistical analyses were performed using the S.A.S. statistics software package (S.A.S. Institute, Cary, NC, 1989).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Evolution of hobo T-lines
Noncompetitive Genetic Background
hobo 3T-lines. The evolution of hobo 3T-lines with regard to the intraGD (intra-strain GD sterility), the activity (induced GD sterility), and the mean copy number of full-sized hobo elements is shown in figure 1. Two situations can be described, according to which parameter is under study. The first situation consists of 3-A and 3-B T-lines showing very low or zero levels of intraGD and activity. In this case, the mean copy number of full-sized elements per fly is estimated to be less than one per individual. The fact that no amplification of the copy number of full-sized elements occurred in 3-A and 3-B T-lines suggests that no invasion occurred in these lines. The second situation involved 3-C and 3-D T-lines that reached high levels of intraGD at the 17th generation (respectively, 18.3% and 15.8 % GD), with an increasing activity between the 5th and the 20th generations (respectively, 10.8% to 35.3% GD and 19.8% to 41.5% GD). The mean copy number of full-sized hobo elements per fly reached approximately five copies at the 20th generation. In the 3-C and 3-D lines there was no correlation between the activity and the mean copy number of full-sized hobo elements (r = 0.42, P > 0.05). The introduction of 3-A and 3-B lines in this analysis leads us to reveal a correlation that is an artifact insofar as two groups are formed; the 3-A and 3-B lines show very low or zero activity with low mean copy number, and the 3-C and 3-D are higher with regard to both parameters.



View larger version (17K):
[in this window]
[in a new window]
 
FIG. 1. Evolution of the 3TPE hobo element in a noncompetitive genetic background. The generations are presented on the abscissa. The GD percentage is in the first ordinate and the mean copy number of full-sized elements (mcn) is in the second ordinate. The activity (GD) was estimated on two replicates of a mean of 50 females per line ({blacksquare}), and the mean copy number was estimated on 30 individuals per line ({diamondsuit}). The intra-strain GD sterility ({square}) was estimated on a mean of 50 females

 
hobo 5T-lines. The evolution of hobo 5T-lines with regard to the intraGD, the activity, and the mean copy number of full-sized hobo elements is shown in figure 2. In hobo 5T-lines, only one situation can be described with regard to all the parameters measured. Indeed, intraGD levels (<10.0% GD on all replicates and all generations) and activity levels (a mean of 7.7% GD on all replicates and all generations) were low apart from the 20th generation of two lines. In 5-C T-line the activity level spectacularly increased between the 17th and the 20th (3.6% to 28.0% GD) generations; and in the 5-D T-line it increased slightly between the 14th and the 20th generations (9.5% to 18.7% GD). No obvious trend in activity evolution is detected in the 5-A and 5-B lines. The mean copy number of full-sized hobo elements in hobo 5T-lines reached a mean of seven copies per fly. No obvious trend in the evolution of the mean copy number of full-sized elements was detected. However, we did notice a spectacular increase in the 5-C line that reached an estimated 29.6 copies at the 20th generation. A significant correlation between the hobo activity and the mean copy number of full-sized elements was detected in hobo 5T-lines (r = 0.44, P < 0.05).



View larger version (19K):
[in this window]
[in a new window]
 
FIG. 2. Evolution of the 5TPE hobo element in a noncompetitive genetic background. The generations are presented on the abscissa. The GD percentage is in the first ordinate and the mean copy number of full-sized elements (mcn) is in the second ordinate. The activity (GD) was estimated on two replicates of a mean of 50 females per line ({blacksquare}), and the mean copy number was estimated on 30 individuals per line ({diamondsuit}). The intra-strain GD sterility ({square}) was estimated on a mean of 50 females

 
hobo 5T-lines versus hobo 3T-lines. With regard to the hobo activity levels, the ANOVA did not reveal a significant effect of the type of line, so there is no difference between hobo 5T-lines and hobo 3T-lines (P > 0.05; table 2). The mean activity level of hobo 5T-lines, estimated from all replicates and all generations, was equivalent to that of hobo 3T-lines (9.1% versus 12.6%). A strong replicate effect was revealed by the ANOVA that is probably due to the noninvasive state in 3-A and 3-C lines (P < 0.001; table 2). In other respects, the ANOVA on the mean copy number of full-sized elements revealed an effect of the type of line (P < 0.01; table 3). The mean copy numbers of full-sized elements, estimated to be 2.2 in hobo 3T-lines and 7.1 in hobo 5T-lines, were significantly different. A replicate effect was revealed that is probably due to the spectacular increase in the mean copy number of full-sized elements in 5-C line (P < 0.05; table 3).


View this table:
[in this window]
[in a new window]
 
Table 2 Hierarchical ANOVA and Duncan Multiple Pairwise Comparison Performed on Arcsin Transformed Data from hobo Activity as Function of the Type of Line and the Generation.

 

View this table:
[in this window]
[in a new window]
 
Table 3 ANOVA and Duncan Multiple Pairwise Comparison Performed on the Mean Copy Number of Full-Sized hobo Elements as Function of the Type of Line and the Generation.

 
Competitive Genetic Background
hobo 35T-lines. The evolution of hobo 35T-lines with regard to the intraGD, the activity, and the mean copy number of full-sized hobo elements is shown in figure 3. With regard to the intraGD levels, two situations can be described in hobo 35T-lines (fig. 3). In 35-A and 35-C T-lines, the levels of intraGD reached higher levels (11.6% and 15.0% GD) than in 35-D and 35-B T-lines (4.2% and 3.0% GD). With regard to the activity levels, the 35-C line is different from other lines, insofar as its level is higher in the first generations tested (23.3% GD versus a mean of 2.8% GD). No obvious trend can be detected in either the evolution of the activity or the evolution of the mean copy number per fly. A significant correlation between activity and mean copy number was detected in hobo 35T-lines (r = 0.44; P < 0.05).



View larger version (19K):
[in this window]
[in a new window]
 
FIG. 3. Evolution of the 3TPE and 5TPE hobo elements in hobo 35T-lines with a competitive genetic background. The generations are presented on the abscissa. The GD percentage is in the first ordinate and the mean copy number of full-sized elements (mcn) is given on the second ordinate. The activity (GD) was estimated on two replicates of a mean of 50 females per line ({blacksquare}), and the mean copy number was estimated on 30 individuals per line ({diamondsuit}). The intra-strain GD sterility ({square}) was estimated on a mean of 50 females

 
hobo 53T-lines. The evolution of hobo 53T-lines with regard to the intraGD, the activity, and the mean copy number of full-sized hobo elements is shown in figure 4. In the hobo 53T-lines, the levels of intraGD are very similar between the lines. With regard to the activity levels, the 53-C and 53-D lines have higher levels than the 53-B and 53-A lines. However, no obvious trends can be observed in either the evolution of the activity or in the mean copy number per fly. No correlation between the activity and the mean copy number was detected in hobo 53T-lines (r = –0.24; P > 0.05).



View larger version (19K):
[in this window]
[in a new window]
 
FIG. 4. Evolution of the 3TPE and 5TPE hobo elements in hobo 53T-lines with a competitive genetic background. The generations are presented on the abscissa. The GD percentage is in the first ordinate and the mean copy number of full-sized elements (mcn) is in the second ordinate. The activity (GD) was estimated on two replicates of a mean of 50 females per line ({blacksquare}), and the mean copy number was estimated on 30 individuals per line ({diamondsuit}). The intra-strain GD sterility ({square}) was estimated on a mean of 50 females

 
hobo 35T-lines versus hobo 53T-lines. In competitive situations, the mean activity levels were estimated to be 14.1% in hobo 35T-lines (on all replicates and at all generations) and 12.8% in hobo 53T-lines and were not statistically different according to the ANOVA (no significant type of line effect; table 2). Hence, with regard to the mean copy number of full-sized elements, estimated to be 4.0 in hobo 35T-lines and 4.4 in hobo 53T-lines, the ANOVA and the Duncan multiple pairwise comparison revealed that the lines were not different (P > 0.05; table 3).

Noncompetitive versus Competitive Genetic Backgrounds
The ANOVA on activity data did not reveal significant differences in the mean activity levels estimated on all replicates and at all generations between hobo 35T-lines / hobo 53T-lines and hobo 5T-lines / hobo 3T-lines (P > 0.05). The ANOVA and Duncan multiple pairwise comparisons revealed that, even though the activity level did not increase through time, it has fluctuated (P < 0.05; table 2). Hence, the activity level at the 5th generation is lower than that of the later generations tested (table 2). With regard to the mean copy number of full-sized elements, the ANOVA revealed a weak replicate effect (P < 0.05; table 3). It also revealed an effect of the type of line (P < 0.01). The Duncan multiple pairwise comparisons revealed that hobo 3T-lines are statistically different from the hobo 53T, hobo 35T, and hobo 5T-lines (P < 0.05). Finally, the ANOVA revealed a generation effect (P < 0.001) that is due to the difference in the mean copy number of full-sized elements between the 5th generation and the 20th generation (Duncan multiple pairwise comparison, P < 0.05).

Permissivity of the Females of the hobo T-lines
The mean level of permissivity of the yw strain is estimated to be 27.81% GD (table 4). The mean level of permissivity of the hobo 3T-lines is estimated to be 45.27% GD, whereas that of the hobo 5T-lines is estimated to be lower, 6.69% GD (table 4). The hobo 3T-lines are significantly more permissive than the yw strain ({chi}2 = 90.82; df = 1; P < 0.001), whereas the hobo 5T-lines are less permissive than this same reference strain ({chi}2 = 204.97; df = 1; P < 0.001). Five of the six lines tested showed lower levels (4.9% to 13.4% GD) than yw, whereas the 53–10 line showed a higher level of permissivity (43.7% GD). Lastly, we observed low levels of permissivity in the F1 progeny of crosses in both directions between a hobo 3T-line and hobo 5T-line (9.5% and 15.2% GD). The variability between the lines bearing both elements could be due to differing proportions of 5TPE and 3TPE elements in these lines.


View this table:
[in this window]
[in a new window]
 
Table 4 Permissivity of the hobo T-lines and yw Females Tested with Males from the 23.5/Cy MRF Strain.

 
Persistence/Loss of hobo Elements in hobo T-lines
To estimate the polymorphism in hobo T-lines, we surveyed the persistence or loss of 5TPE or 3TPE hobo elements in individuals. Then, the distribution of the hobo elements in the 16 hobo T-line individuals was analyzed in samples of 10 flies per line by an h11-h6 PCR amplification at the 7th and 20th generations. For each type of line, we compared the replicates at the 7th and 20th generations, and then we compared these generations to detect the change over time. We also compared the hobo 5T-lines and hobo 3T-lines, and then the lines with a competitive genetic background (hobo 35T-lines plus hobo 53T-lines) to each type of noncompetitive line (hobo 3T-lines and hobo 5T-lines). Among 24 comparisons (exact Fisher tests) only three were significant after correction using the sequential Bonferroni technique.

hobo 3T-lines versus hobo 5T-lines
In hobo 3T-lines and hobo 5T-lines, two classes were expected: individuals harboring hobo elements ([3] or [5] class) and individuals devoid of hobo elements ([E] class) (table 5). All the Fisher exact tests that we performed in hobo 3T-lines and hobo 5T-lines were nonsignificant (P > 0.05), suggesting that the replicates in hobo 3T-lines and hobo 5T-lines were homogeneous in any given generation and that no change in the proportions occurred over time. The hobo 3T-lines and hobo 5T-lines were not significantly different at the 7th generation tested with regard to the proportions of [H] individuals (i.e., [3] or [5]) and [E] individuals (P > 0.05). In contrast, hobo 3T-lines and hobo 5T-lines were significantly different at the 20th generation (P < 0.05). This difference is due mainly to the under-representation of the [3] class in the 3-B line.


View this table:
[in this window]
[in a new window]
 
Table 5 Proportions of the Different Classes of Individuals Intra Lines.

 
Noncompetitive Genetic Background versus Competitive Genetic Background
The proportions of the different classes of individuals in hobo 35T-lines and hobo 53T-lines are shown in table 5. Four classes [3] (3TPE element), [5] (5 TPE element), [3,5] (3 and 5 TPE elements), and [E] (empty) were expected. With regard to these expected classes in the hobo 35T-lines and hobo 53 T-lines, all the statistical analyses that we performed were nonsignificant (P > 0.05) apart from the 20th generation in hobo 53T-lines (P < 0.05). This difference is probably due to the total lack of [3] and [3,5] class individuals in the 53-B line (table 5). Despite this result, the maternal origin of the elements did not give rise to any difference of behavior in the early generations. Indeed, at the 7th generation, the [3] class and [5] class were equally represented in the hobo 35T-lines and hobo 53T-lines. It was striking that at the 20th generation, the [3] class was under-represented relative to the [5] class. In a competitive situation, the 3TPE element could be less efficiently preserved alone in the [3] class than the 5TPE element in the [5] class. However, the 3TPE element did not disappear from the lines as it was maintained in the [3,5] class. To compare the dynamics of the 3TPE element in competitive and noncompetitive genetic backgrounds, we created two classes similar to those for the hobo 3T-lines (table 5). In the hobo 35T-lines and hobo 53T-lines, the [3] and [3,5] classes are summed to constitute the [3,*] class (individuals bearing the 3TPE element), and the [5] and [E] classes are summed to constitute the [E/3] class (individuals empty of the 3TPE element). We performed the same comparison between hobo 35T-lines and 53T-lines with regard to the 5TPE element. In this case, the [5] and [3,5] classes were summed to constitute the [5,*] class, and the [3] and [E] classes are summed to constitute the [E/5] class.

These comparisons revealed just one significant difference at the 7th generation between the [5,*] and [E/5] classes of the hobo 35T-lines plus hobo 53T-lines and the [5] and [E] classes of hobo 5T-lines (P < 0.05). Such a difference was not found when we compared [3,*] and [E/3] classes to [3] and [E] classes in hobo 3T-lines. The initiation of each hobo 3T-line and hobo 5T-line involved one pair of flies that were heterozygous for the insert containing the hobo element. The initiation of each hobo 35T-line and hobo 53T-line involved one pair of flies heterozygous for one insert (containing the 3TPE element or 5TPE element) and homozygous for the site without the other insert. Thus, in early generations the [E/3] and [E/5] classes in the hobo 35T-lines and hobo 53T-lines were expected to be higher than the [E] classes in the hobo 3T-lines and hobo 5T-lines. Unexpectedly, the hobo 35T-lines and hobo 53T-lines did not differ from hobo 3T-lines ([3,*], [E/3] vs. [3], [E]), whereas such a difference was detected with regard to the 5TPE hobo element. Then, in a competitive genetic background, the 3TPE hobo element could be more invasive than the 5TPE element in early generations.

Deleted Elements
Deleted elements of very similar sizes (1.8 kb XhoI fragment) were detected in some lines from each type at different times during the evolution of the lines. Curiously, deleted elements were detected in the 3-A and 3-B lines from the 5th generation, revealing that at least one excision and DNA repair event had occurred before this generation (fig. 5). Deleted elements (1.8 kb XhoI fragment) were also detected in the 5-A line later than in the hobo 3T-lines, at the 17th and the 20th generations (data not shown). The very same elements were also detected in the 35-A T-line from the 5th generation (fig. 5), and later in the 35-D T-line at the 11th generation (data not shown). Finally, they were detected in the 53-A line at the 11th and 20th generations (data not shown).



View larger version (74K):
[in this window]
[in a new window]
 
FIG. 5. Detection of full-sized and deleted elements at the 5th generation of hobo L-lines. Southern blot first hybridized with a white gene probe (upper image) and then with a hobo108 probe. Bands revealed by the white gene probe area as follows: {blacktriangleleft} endogenous white gene; {blacktriangleright} supernumerary bands corresponding to the initial transgene. Bands revealed by the hobo108 probe correspond to the arrows, as follows: <- 2.6 kb XhoI internal fragment of full-sized hobo elements; <-1.8 kb XhoI fragment of deleted elements. High molecular weight bands (>2.6 kb) correspond to the heterochromatic degenerated hobo sequences (Daniels, Chovnick, and Boussy 1990). yw is a strain devoid of hobo elements. CyHBL1 is a strain containing one copy of the HFL1 element per diploid genome

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Most of the hobo elements detected in natural populations of D. melanogaster are present at very low frequencies, with the exception of the 3TPE, 4TPE, and 5TPE elements. Moreover, the 3TPE element is distributed worldwide, whereas the 4TPE and 5TPE elements have narrower distribution areas (Bonnivard, Bazin, and Higuet 2002). These surveys of natural populations suggest that the different hobo elements could have different invasive potentials, depending on the number of TPE repeats. To study the invasive ability of 5TPE hobo elements, first alone in the context of a noncompetitive genetic background and then in a competitive genetic background, we established hobo T-lines of D. melanogaster that bore the 3TPE element and/or the 5TPE element. We investigated these lines during the first 20 generations.

The GD sterility is the symptom of the hybrid dysgenesis syndrome that we used to assess the hobo activity level, the permissivity level, and the result of both parameters i.e., the intra-strain GD sterility. This raises the question of whether the GD sterility is due to hobo elements or to an inbreeding effect in the host strain. We can clearly attribute the GD sterility to the presence of hobo elements insofar as neither the yw host strain nor the zola test strain ever shows any GD sterility at 25°C (complete agonadism). Moreover, both reciprocal crosses between individuals from the yw strain and the zola strain never show any GD sterility in the same conditions.

In our experiment the mean copy number of hobo elements per fly remained low (from ~4 to 7), as in previous studies of the 3TPE element (from 2 to 10) carried out by Ladevèze et al. (1994, 1998, 2001) and Galindo et al. (1995). In some of our lines, XhoI fragments of the same size (~1.8 kb), putatively corresponding to 2.1 kb internally deleted elements (deletion of 800 bp), appeared independently in the lines at different generations. These deleted elements are bigger than the Th and Oh elements consisting of 1.5 and 1.1 kb XhoI fragments, respectively (Périquet et al. 1989), and the 1.7 kb Kh element (Kim and Kim 1999). It would be interesting to clone these deleted elements to see whether they have all been deleted from the same sites. There is no evidence that these deleted elements are involved in regulating the activity of the hobo elements.

The lines that bore only the 3TPE element (hobo 3T-lines) are clearly different from the other lines we established. Indeed, hobo 3T-lines are particularly distinct insofar as two situations were observed. In two lines (3-A and 3-B), the introduction of a hobo element did not trigger an invasion of the genome, whereas in the other two (3-C and 3-D) the element did start to invade. In contrast, the introduction of the 5TPE element triggered an invasion in all the hobo 5T-lines. The heterogeneity of hobo 3T-lines could be explained by a position effect of the initial insert. However, the progenitor transgenic line of 3-A and 3-B lines harbored two inserts, and when established, the 3-A line inherited both inserts whereas the 3-B line inherited only one (fig. 5). Even if one of the initial inserts was locked in a specific position on the genome, it is unlikely that both inserts would be. However, because we started our analyses at the 5th generation, it is not impossible that the 3TPE element was active in these lines. This would be consistent with the fact that deleted elements appeared from these lines insofar as the appearance of such elements is related to the excision of the complete element and the DNA repair step that follows. Lastly, these two lines raised an interesting issue in that despite the noninvasive state, the element is maintained in the lines. Moreover, none of the 26 hobo 3T-lines initially established have lost their elements as revealed by an h11-h6 PCR analysis on samples of 10 mixed flies at the 16th generation.

The hobo 5T-lines are more homogeneous than hobo 3T-lines. Moreover, the mean copy number of full-sized elements is higher in hobo 5T-lines than in hobo 3T-lines, but the activity levels were equivalent. This suggests that the 5TPE element is less active than the 3TPE element in a noncompetitive genetic context. This could explain why, in hobo 5T-lines, unlike the hobo 3T-lines, we found a correlation between the activity and the mean copy number of full-sized elements. Indeed, if the activity of the element is low; the copy number would have an impact on the level of activity, whereas this would not be the case if the element was very active—i.e., one copy inducing a high level of activity. Moreover, with regard to permissivity, females bearing 3TPE elements showed a higher level of permissivity than females from the yw strain. In contrast, females bearing 5TPE elements showed a lower level of permissivity than females from the yw strain or from hobo 3T-lines. This suggests that the 3TPE element could induce GD sterility in the nondysgenic cross. However, the 3–28 line shows no GD sterility when crossed either way with the yw strain or when crossed with 23.5/Cy MRF females (data not shown). This suggests that the 3TPE element could enhance the activity of the 3TPE elements of the 23.5 MRF chromosome, whereas the 5TPE element could regulate the activity of these elements.

The proportions of the different classes of individuals in the lines with regard to the presence of 3TPE and/or 5TPE elements showed that the hobo 5T-lines seem to maintain their elements at least as efficiently as the hobo 3T-lines. However, in a competitive genetic background, we observed that there were relatively fewer individuals belonging to the [3] class than to the [5] class, despite an equilibrated situation at the 7th generation. This could suggest that in a competitive situation the 5TPE element may be more efficiently maintained alone (the [5] class) than the 3TPE element alone (the [3] class). This situation does not mean that the 3TPE element will necessarily disappear, as it could be maintained, mainly in the [3,5] class. This situation can be extended to the 87 hobo T-lines remaining from the 100 lines initially established. These lines were studied by an h11-h6 PCR on samples of 10 mixed flies per line. At the 16th generation, all the lines that initially had the 3TPE element or the 5TPE element gave a PCR signal. In the 34 lines that initially bore the 3TPE element and the 5TPE element, we observed 18 [3,5] lines, 12 [5] lines, 1 [3] line, and 3 [E] lines. Because of the under-representation of the [3] class relative to the [5] class, we could suggest that in a competitive context, the 3TPE element is maintained better when accompanied by the 5TPE element, whereas the 5TPE element is maintained even if it occurs alone. This could result in a higher frequency of the 5TPE element than of the 3TPE element in a competitive situation, and this would be consistent with the situation seen in some Western European populations that do indeed display higher frequencies of the 5TPE element (Bonnivard, Bazin, and Higuet 2002).

With regard to the hobo activity, the hobo 35T-lines and hobo 53T-lines are not different from the hobo 5T-lines. This suggests that the 5TPE element could decrease the activity of the 3TPE element. This is consistent with the results of the analysis of the permissivity in hobo 3T-lines and hobo 5T-lines that showed that the 5TPE hobo elements regulate the activity of 3TPE elements. Moreover, in hobo 35T-lines and hobo 53T-lines, the levels of permissivity are close to that of hobo 5T-lines. To explain such an effect, we have to speculate about the mechanistic aspects of the hobo element because the structure of the putative transposase of the hobo element is particularly poorly documented. However, Essers, Adolphs, and Kunze (2000) showed that the transposase of the maize Ac transposable element contains a domain involved in dimerization. This domain is highly conserved in the hAT superfamily. In the hobo element, this domain is located at 123 bp downstream from the TPE repeats (41 amino acids at the protein level). We could therefore argue that hobo transposase could at least act as a dimer, and consequently any change in the number of TPE repeats could have an impact either on the dimerization or the presentation of the active site of the transposase. In a noncompetitive situation the hobo transposase can only be a homodimer of 3TPE or 5TPE transposase, but in a competitive situation three classes of transposase could be formed: a 3TPE or 5TPE homodimer or a heterodimer. It is possible that in a competitive situation the 5TPE element could poison the 3TPE element transposase and that this would give it a competitive advantage by decreasing deleterious effects. Such a phenomenon could explain why the 3TPE element is maintained mainly in the [3,5] class in hobo 35T-lines and hobo 53T-lines. We need now to investigate further the hypothesis of the dimeric action of the hobo transposase relative to the TPE repeats, which could be the key to understanding the history of hobo elements in D. melanogaster species.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
We thank Marcela Antivilo, Céline Gomez, and Chantal Labellie for their helpful technical assistance; Monika Ghosh for language revision; and Béatrice Denis for graciously providing plasmids and strains. This work was supported by GDR 2157-CNRS "Evolution des éléments transposables: du génome aux populations."


    Footnotes
 
Pierre Capy, Associate Editor Back


    Literature Cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 

    Bazin, C., and D. Higuet. 1996. Lack of correlation between dysgenic traits in the hobo system of hybrid dysgenesis in Drosophila melanogaster. Genet. Res. 67:219-226.[ISI][Medline]

    Blackman, R. K., R. Grimaila, M. M. Koehler, and W. M. Gelbart. 1987. Mobilization of hobo elements residing within the decapentaplegic gene complex: suggestion of a new hybrid dysgenesis system in Drosophila melanogaster. Cell. 49:497-505.[ISI][Medline]

    Bonnivard, E., C. Bazin, B. Denis, and D. Higuet. 2000. A scenario for the hobo transposable element invasion, deduced from the structure of natural populations of Drosophila melanogaster using tandem TPE repeats. Genet. Res. 75:13-23.[CrossRef][ISI][Medline]

    Bonnivard, E., C. Bazin, and D. Higuet. 2002. High polymorphism of TPE repeats within natural populations of D. melanogaster: a gradient of the 5TPE hobo element in Western Europe. Mol. Biol. Evol. 19:2277-2284.[Abstract/Free Full Text]

    Brand, A., and N. Perrimon. 1993. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 118:401-415.[Abstract/Free Full Text]

    Calvi, B. R., and W. M. Gelbart. 1994. The basis for germline specificity of the hobo transposable element in Drosophila melanogaster. EMBO J. 13:1636-1644.[Abstract]

    Calvi, B. R., T. J. Hong, and W. M. Gelbart. 1991. Evidence for a common evolutionary origin of inverted repeat transposons in Drosophila and plants: hobo, Activator, and Tam3. Cell. 66:465-471.[ISI][Medline]

    Daniels, S. B., A. Chovnick, and I. A. Boussy. 1990. Distribution of hobo transposable elements in the genus Drosophila. Mol. Biol. Evol. 7:589-606.[Abstract]

    DiFranco, C., A. Terrinoni, D. Galuppi, and N. Junakovic. 1995. DNA extraction from Drosophila individual flies. Drosophila Information Service. 76:172-174.

    Essers, L., R. H. Adolphs, and R. Kunze. 2000. A highly conserved domain of the maize activator transposase is involved in dimerization. Plant Cell. 12:211-224.[Abstract/Free Full Text]

    Galindo, M. I., V. Ladevèze, F. Lemeunier, R. Kalmes, G. Périquet, and L. Pascual. 1995. Spread of the autonomous transposable element hobo in the genome of Drosophila melanogaster. Mol. Biol. Evol. 12:723-734.[Abstract]

    Junakovic, N., R. Caneva, and P. Ballario. 1984. Genomic distribution of copia-like element in laboratory stocks of Drosophila melanogaster. Chromosoma. 90:378-382.[ISI]

    Kidwell, M. G., and J. B. Novy. 1979. Hybrid dysgenesis in Drosophila melanogaster: sterility resulting from gonadal dysgenesis in the P-M system. Genetics. 92:1127-1140.[Abstract/Free Full Text]

    Kim, J. M., and W. Kim. 1999. Identification of a full-size hobo element and deletion-derivatives in Korean populations of Drosophila melanogaster. Mol. Cells 9:127-132.[ISI][Medline]

    Ladevèze, V., S. Aulard, N. Chaminade, C. Biemont, G. Périquet, and F. Lemeunier. 2001. Dynamics of the hobo transposable element in transgenic lines of Drosophila melanogaster. Genet. Res. 77:135-142.[CrossRef][ISI][Medline]

    Ladevèze, V., M. I. Galindo, N. Chaminade, L. Pascual, G. Périquet, and F. Lemeunier. 1998. Transmission pattern of hobo transposable element in transgenic lines of Drosophila melanogaster. Genet. Res. 71:97-107.[CrossRef][ISI][Medline]

    Ladevèze, V., M. I. Galindo, L. Pascual, G. Périquet, and F. Lemeunier. 1994. Invasion of the hobo transposable element studied by in situ hybridization on polytene chromosomes of Drosophila melanogaster. Genetica. 93:91-100.[ISI][Medline]

    Laski, F. A., D. C. Rio, and G. M. Rubin. 1986. Tissue specificity of Drosophila P element transposition is regulated at the level of mRNA splicing. Cell 44:7-19.[ISI][Medline]

    Périquet, G., M. H. Hamelin, Y. Bigot, and Kai Hu. 1989. Presence of the deleted elements Th in Eurasian populations of Drosophila melanogaster. Genet. Sel. Evol. 21:107-111.[ISI]

    Rice, W. R. 1989. Analyzing tables of statistical tests. Evolution 43:223-225.[ISI]

    Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual. Cold Spring Laboratory Press, Cold Spring Harbor, NY.

    Souames, S., E. Bonnivard, C. Bazin, and D. Higuet. 2003. High mutation rate of TPE repeats: a microsatellite in the putative transposase of the hobo element in Drosophila melanogaster. Mol. Biol. Evol. (in press).

    Stamatis, N., M. Monastirioti, G. Yannopoulos, and C. Louis. 1989. The P-M and 23.5 MRF (hobo) systems of hybrid dysgenesis in Drosophila melanogaster are independent of each other. Genetics 123:379-387.[Abstract/Free Full Text]

    Streck, R. D., J. E. MacGaffey, and S. K. Beckendorf. 1986. The structure of hobo element and their insertion site. EMBO J. 5:3615-3623.[ISI]

    Yannopoulos, G., N. Stamatis, A. Zacharopoulou, and M. Pelecanos. 1983. Site-specific breaks induced by the male recombination factor 23.5 MRF in Drosophila melanogaster. Mutat. Res. 108:185-202.[ISI][Medline]

    Yannopoulos, G., N. Stamatis, M. Monastirioti, and C. Louis. 1987. hobo is responsible for the induction of hybrid dysgenesis by strains of Drosophila melanogaster bearing the male recombination factor 23.5 MRF. Cell 49:487-495.[ISI][Medline]

Accepted for publication July 14, 2003.





This Article
Abstract
FREE Full Text (PDF)
All Versions of this Article:
20/12/2055    most recent
msg221v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (2)
Request Permissions
Google Scholar
Articles by Souames, S.
Articles by Higuet, D.
PubMed
PubMed Citation
Articles by Souames, S.
Articles by Higuet, D.