Decreased levels of dCAP-D3/Condensin II result in a local loss of retrotransposon sequence <i>in vivo</i> and <i>in vitro</i>.

<p>A) PCR performed on DNA from wild type (<i>w¹¹¹⁸</i>) adults and dCAP-D3 transheterozygous mutant adults (d<i>Cap-D3^c07081</i>d<i>Cap-D3^/Δ25</i>) to detect whether three different retrotransposon sequences (<i>mdg1-1403, G2-1077, X-978</i>) were present (P) or absent (A) in dCAP-D3 regulated gene clusters indicates absence of the retrotransposon sequence only in <i>dCap-D3</i> mutants. In the diagram for each locus, transposon positions are illustrated with pink brackets. Single black boxes represent the entire coding sequence for each gene, except in the case of CG42335 where two black boxes are used to represent the coding sequence upstream and downstream of the <i>mdg1-1403</i> retrotransposon. Primers sets used are depicted in the diagrams above the gels and their sequences can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003879#s4" target="_blank">Materials and Methods</a>. Tubulin23C (Tub) was used as a control for each reaction. In the PCRs performed on the <i>mdg1-1403</i> locus, an asterisk denotes the band for presence. The miscellaneous band seen in the wild type absence reaction was confirmed to be a mispriming event off of tubulin (data not shown). B) PCR performed to detect presence or absence of <i>mdg1-1403</i> in different <i>dCap-D3</i> mutant genotypes shows that events which cause local loss of retrotransposon sequence increase as dCAP-D3 expression levels decrease. PCR was performed in individual, female, wild type flies and flies expressing different <i>dCap-D3</i> mutant alleles. <i>dCap-D3</i> mutants were heterozygous or homozygous for a hypomorphic allele of <i>dCap-D3</i> (<i>c07081</i>) which expresses about 10% the levels of wild type protein <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003879#pgen.1003879-Longworth2" target="_blank">[8]</a>, or transheterozygous for a combination of the hypomorphic allele and a deletion (<i>c07081/Δ25</i>). <i>rp49</i> was used as a control for each reaction. C) PCRs performed as described in (A) on DNA from flies expressing two mutant alleles of a second Condensin II subunit, <i>dCap-H2</i> (<i>dCap-H2^Z3-0019/dCap-H2^Z3-</i>⁵¹⁶³), demonstrates identical results seen for <i>dCap-D3</i> mutants. D) PCR for <i>mdg1-1403</i> presence or absence in Condensin I subunit, <i>dCap-D2</i>, heterozygous mutants <i>(dCap-D2^f03381/+)</i> shows only presence of the retrotransposon. PCRs were performed on 1) wild type adults to test for presence, 2) wild type adults to test for absence, 3) <i>dCap-D2</i> mutants to test for presence and 4) <i>dCap-D2</i> mutants to test for absence. E) PCR for presence and absence of <i>mdg1-1403</i> (left) and <i>G2-1077</i> (right) in SG4 cells treated with control or dCAP-D3 dsRNAs for 6 days shows the appearance of an absence band (pink arrows) in dCAP-D3 dsRNA treated cells but not in control dsRNA treated cells.