We have screened 1375 2nd and 3rd chromosome P-element lethal lines from both Bloomington and Szeged Stock Centers for this study. Most of the 779 Bloomington lines have sequence information on the flanking genomic region of each P-element insertion, whereas most of the 596 Szeged lines have no flanking sequence information available (1). Components of a modified FLP/FRT system were introduced into the P-element stocks (Figure S1; 2,3) to produce homozygous (P[w+] / P[w+]) mutant eyes in a heterozygous and viable animal.
Figure S1. A five-generation crossing scheme for the Bloomington P-element lines on the 3L chromosomal arm. Similar schemes were also used for P-element lines on the 2L, 2R and 3R arms. The first (P) cross introduces ey-Flp, while the second (F1) cross generates a P[w+] / FRT heterozygous genotype in the females so that meiotic recombination can take place in her gametes. Among their progeny (F2) are the "small clone" mosaic flies that inherit the P[w+] FRT recombinant chromosome and can be unambiguously selected based on the unique orange-white mosaic eyes as a result of the ey-Flp driven mitotic recombination in the early eye discs (see Fig S2). Four independent crosses were carried out between one male that had a "small clone" mosaic eye and multiple FRT w+M females (F3) to yield two types of progeny: the heterozygous P[w+] FRT with a third chromosome balancer TM6B,y+, in which male and female siblings were crossed to establish a balanced stock of the recombinants (F4), and the mosaic flies that display a large (usually ~90% of the eyes) homozygous P[w+]/P[w+] clone due to the presence of the Minute dominant mutation. The "large clone" mosaic eyes facilitate the screening process and were used for the determination of eye phenotype.
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To accomplish this, we took advantage of the presence of the mini-w+ transgene (a truncated form of the wild type white (w) gene that contributes to the fly's eye color) in the P-elements. Wild type fly eyes are dark-red in color, while a recessive mutation of the w gene causes white eye color. The mini-w+ transgene in the P-element usually confers yellow or orange color to adult Drosophila eyes in a mutant w genetic background, and these lighter colors are crucial to the identification of mosaic eyes later in the process (Figure S2B).
Figure S2 A) Generation of small mutant clones in the eye. Flies that have undergone meiotic recombination between the FRT site and the P-element have white and orange mosaic eyes when performing small clone analysis. The ability to unambiguously select for these flies with these mosaic eyes was the main purpose for generating small clones in our screen. B) Generation of large mutant clones in the eye. As in the small clone, ey-Flp mediated mitotic recombination was utilized, but now in the presence of a lethal Minute (M) mutation. This M chromosome was marked by a dark red eye. By using this system, very large homozygous mutant clones were made that were orange, while the heterozygous tissue was dark red.
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Through 5 generations of genetic crosses (Fig S1), each of the 779 Bloomington P-element mutations were meiotically recombined onto an FRT containing chromosome (FRT40A for 2L, FRT42D for 2R, FRT80B for 3L and FRT82B for 3R). The 596 Szeged P-element lines already have an FRT on each P-element mutant chromosome and did not need to go through the whole crossing scheme (4). Concurrently, an X chromosome that contains a construct expressing Flippase under the control of eyeless enhancer was introduced into the flies. Flippase is expressed exclusively in the early eye progenitor cells, resulting in mitotic recombination (Figure S2 A,B; 3). In the end, a total of 1375 FRT-P[w+] lines were crossed to flies containing FRT w+ Minute to generate the "large clone" mosaics in the next generation. Minutes (M) can be found on each chromosome and encode for ribosomal components. Cells heterozygous for M have slower proliferation and cells homozygous for M die. The addition of M to the wild type FRT chromosome causes large homozygous mutant clones that usually constitute ~90% of the adult eyes, allowing for a more sensitive screen for eye phenotypes (Figure S2B, 3).
Light microscopy (Nikon E600 with a Nikon Coolpix 4500 camera) was performed on all the "large-clone" mosaic eyes. In addition, Scanning Electron Microscopy (SEM; Hitachi 2460N) images were taken for flies with any mutant eye phenotype. 499 mutants were isolated and could be grouped into three major classes: cell lethal, rough and glossy. These major individual mutant phenotypes have been classified in the database. Also noted are the mutants that exhibited combinations of the aforementioned phenotypes. For a small number of mutants, no adults could be recovered, and they were thus classified as "adult non-recoverable".
Verification and Excision Experiments
All 1375 FRT-P[w+] lines were re-crossed to FRT / FRT or FRT w+ M / Balancer stocks to verify the previously determined small-clone and large-clone phenotypes. For 294 of the eye mutants, the FRT-P[w+] stocks were crossed to flies expressing the Transposase (delta 2-3) in order to excise the P-element out of the genome and to determine if the P-element is responsible for the mutant eye phenotype (Figure S3).
Figure S3. Crossing scheme of the P-element excision experiment on the 3L chromosomal arm. Similar schemes were also used for excision of P-elements on the 2L, 2R and 3R arms. FRT-P element lines were crossed with a flies expressing transposase (delta 2-3) to cause the excision of the lethal P-element. If the original P-element insertion was responsible for the phenotype, excision of the P-element would result in a fly that has wild-type eyes that are predominantly white.
If the P-element insertion is responsible for the eye phenotype, precise excision of the P[w+] would result in the eyes being completely wild type. For the purposes of standardizing our results, if there were no wild type revertants observed in 1000 progeny scored, then we called that a failed excision. We observe that 72% of the eye mutants revert back to wild type after excision of the P-element, comparable to the 65% reversion rate that was reported in a recent study on X chromosome lethal P-element mutations (5).
Determination of the BLAST hit, gene disrupted and gene function categories
Flanking sequence from inverse PCR previously performed by other laboratories was obtained from the FlyBase (www.flybase.net). The flanking sequence was used to determine the genomic insertion site. In the database, a gene is listed as "disrupted gene 1" if the P-element insertion is inserted in the coding region, intron, or within 3 kb of the 5' end of that gene. If a second gene is close to the insertion site, it is designated "disrupted gene 2". If no gene could be found within 3 kb, the insertion is designated as NG (no gene). Functional categories were only created for "disrupted gene 1" hits by reviewing the molecular function, biological process and protein domains found in the FlyBase database. Note that the category designation is based on the Drosophila Genome Annotation Release 3.2.1 and may evolve over time.
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