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Cart 0 items. Toggle navigation. Note: Cover may not represent actual copy or condition available. Brand New. No shipping to PO Add to cart. Territorial restrictions may be printed on the book. For expedited shipping, Get it fast This could be explained by an ongoing transposition at cold conditions, which generated novel low-frequency insertions, compensating for the loss of low-frequency insertions by drift. Because all base substitutions within the P-element were at low frequencies Supplemental Results S1 , the P-element did not evolve during the invasion.
The P-element had a similar insertion bias in all replicates Supplemental Results S2. The difference in the invasion dynamics under hot and cold conditions Fig. This may account for the slower invasion under cold conditions, although we cannot rule other factors, such as temperature-dependent efficiency of the transposase. Furthermore, it is not clear to what extent P-element expression in whole bodies and ovaries is correlated.
To test whether the P-element is transcribed in the ovarian germline, we performed single-molecule RNA-FISH, using ovaries dissected from flies of the hot invasion at generation and from the cold invasion at generation The presence of P-element transcripts sense in nurse cells Fig. Production of a functional transposase requires splicing of the third intron, which is restricted to the germline. Despite many more reads mapping to the P-element , we found no evidence for splicing of the third intron in hot-evolved populations at generation 22 Supplemental Table S4.
This suggests that a functional P-element transposase is generated in cold-evolved populations at generation 11 but not in hot-evolved populations at generation 22, which is consistent with plateauing of the hot invasion by generation 20 Fig. P-element expression at generation 22 or later at hot conditions may therefore be mostly somatic, or splicing of the third intron may be suppressed by the piRNA pathway Teixeira et al.
The influence of TE activity on resident TE families has been studied in dysgenic crosses. In some studies, TE activity reactivated dormant families Petrov et al. Dysgenic crosses may not reflect the dynamics during a TE invasion, because they do not yield fertile offspring and novel TE insertions are not passed to the next generation. Only the fraction of reads mapping to the P-element increased during the hot as well as the cold invasion, while the other TE families remained constant Supplemental Fig. We conclude that other TE families are not influenced by the dynamics of the P-element.
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P-elements with internal deletions are common in natural populations and in laboratory strains O'Hare and Rubin ; Kofler et al. Some of the internally deleted elements, such as the KP-element , repress P-element activity Black et al. P-elements with internal deletions express nonfunctional transposases that retain DNA-binding capacity and prevent functional transposases from accessing the transposase-binding sites and thus from mobilizing the P-element Lee et al.
Because of the tight coupling between P-element activity and the generation of internally deleted copies Engels et al. To address this idea, we searched for internally deleted P-elements in the experimentally evolving populations and realigned all reads mapping to the P-element allowing for large gaps.
We identified different internal deletions in hot populations summed over all generations and replicates and 27 in cold populations Fig. Fifteen of the internal deletions were found in more than one of the six experimental populations. The most parsimonious explanation is that these internal deletions were present in the base population. Hence, about internal deletions emerged during the hot invasion and 12 in the cold invasion. Consistent with deletions arising from interruption of sister chromatid—mediated gap repair Engels et al.
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S13 : As gap repair proceeds from both ends of a double-stranded break, interruptions will mostly happen before central parts of the P-element can be replicated. Dynamics of internally deleted P-elements during the invasion. A All internal deletions identified under hot and cold conditions are shown sum over all replicates and generations.
Horizontal bars represent the deleted sequence. The position of the two red internal deletions differs by a single nucleotide, so they likely refer to the same internal deletion. The position of two internal deletions with documented P-element repression KP and D50 are indicated Black et al. Regions required for mobilization of the P-element are shaded in green, and regions required for repressing P-element activity are shaded in orange Majumdar and Rio B An unbiased comparison of the abundance of internally deleted P-elements between hot and cold conditions.
The physical coverage of the P-element has been times randomly sampled to Internal deletions are consistently more frequent in hot-evolved populations. C Frequency of internally deleted P-elements during the hot and cold invasion for all three replicates right panel. The color of the trajectories is as in panel A , allowing identification of the position within the P-element of internal deletions.
D Fitness landscape of internally deleted P-elements : average population frequency of internal deletions covering a given site. A high average frequency indicates that deletion of a site is advantageous and leads, on average, to a frequency increase of an internally deleted P-element. Note that few internal deletions were observed under cold conditions. The higher number of internally deleted P-elements under hot conditions could be an artifact of 1 more advanced generations sequenced, 2 the higher abundance of P-elements , and 3 differences in coverage between samples.
To ensure an unbiased comparison, we analyzed the abundance of internally deleted P-elements at the same generations in the two environments and subsampled the coverage of the P-element to 52 corresponding to randomly sampling 52 P-elements from every sample. There were consistently more internally deleted P-elements under hot than under cold conditions Fig. We found that microhomology extends 2 nt upstream of and downstream from deletion breakpoints, which is consistent with nonhomologous end joining repairing double-stranded breaks resulting from interrupted gap repair Supplemental Fig.
S11 ; Engels et al. P-elements with internal deletions rapidly emerged in our experimental populations. We estimated the frequency of internally deleted P-elements using short read data. By considering all reads mapping to the P-element without inference of the genomic position , the abundance of a given internal deletion relative to the total population of P-elements can be assessed. We found that several internal deletions dramatically increased in frequency during both hot and cold invasions, with some reaching frequencies as high as 6.
For every position in the P-element , we averaged the frequency of all internal deletions covering the site. Fitness landscapes were computed across all replicates using the frequency at generation 60 and 40 for hot- and cold-evolved populations, respectively. The four explanations of the increased frequency of internally deleted P-elements predict distinct fitness landscapes Supplemental Fig. Neutral deletions or deletions linked to positively selected variants will result in a similar average frequency across the entire P-element Supplemental Fig.
In the case of positive selection for internally deleted copies suppressing P-element activity, the DNA-binding domain of the P-element transposase required for repression Fig. If internally deleted P-elements are more readily mobilized than full-length elements Itoh et al. S12C ; Majumdar and Rio Our analysis revealed a low average frequency at sites necessary for mobilization Fig. S13 , implying that the internally deleted P-elements increase in frequency because they are more readily mobilized.
We note, however, that the average frequency estimates at the ends of the P-element are based on few internal deletions and may thus be less reliable than in central regions.
Lee et al. With a frequency of 7. To test this idea, we measured the abundance of piRNAs complementary to the P-element in our experimental populations by sequencing small RNAs at multiple time points Fig. S14, S The dramatic increase in piRNA levels during the cold invasion is not an artifact of normalization because the piRNA abundance of all other TE families is stable during the hot and the cold invasion Supplemental Fig. S16, S It is unlikely that these two peaks are sequencing or mapping artifacts, as neither was found in the cold invasion at generation 22 Fig.
Dynamics of piRNAs during the P-element invasion. Gray triangles indicate the time of sequencing of small RNA libraries. A large peak at position was truncated. The ping-pong signature of D. E Antisense transcripts of the P-element are expressed in D. We dissected flies from the hot invasion Hot at generation and from the cold invasion Cold at generation 54 and used single RNA-FISH to detect antisense transcripts of the P-element black dots. Consistent with observed plateauing of the hot invasion between generation 18 and 20, a functional piRNA-based defense system was established by generation 22 Fig.
As the P-element was not entirely silenced by generation 40 in the cold invasion Figs. The temperature effect may be mediated by different numbers of insertions in the hot and cold invasion resulting in a more rapid silencing of abundant TEs.
Further evidence for the buildup of an active secondary piRNA pathway comes from the observation that the weakest signal of antisense transcripts was found under cold conditions in replicate 5, which has the fewest piRNAs Fig. This suggests that piRNA production requires a sufficient supply of antisense transcripts.
During the ping-pong cycle, RNA cleavage products of Aub are loaded onto Ago3 and vice versa, where the Aub cleavage site is shifted by 10 bp from the Ago3 cleavage site. We found a peak at position 10 for the hot invasion at generations 22, 44, and , and for the cold invasion at generation 54 Fig. S19 ; excluding the antisense peaks at positions and ; Supplemental Fig.