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Figure 4 | Genome Biology

Figure 4

From: Both subtelomeric regions are required and sufficient for specific DNA fragmentation during macronuclear development in Stylonychia lemnae

Figure 4

Functional analysis of the 5' end of the 1.3 kb macronuclear precursor sequence. Different deletion constructs were injected into the developing macronucleus and the cells were allowed to finish macronuclear development. The presence of the injected constructs in the macronuclear DNA and their processing was analyzed by PCR. All constructs were injected into the polytene chromosome stage of the macronuclear anlagen; injection conditions were as described earlier [27]. A PCR analysis was performed using DNA of 30-40 vegetatively growing cells as template. Primers used were P9 and P7, both derived from endogenous 1.3 kb macronuclear sequences, and P548, derived from the inserted polylinker. (a) PCR products from uninjected and injected cells were separated on a 1% agarose gel. As a control a PCR using vector pCE5 as template was performed. M, molecular weight marker (1 kb ladder, Gibco BRL). Lane 1, PCR product from pCE5 vector DNA using the primer combination P9/P548. Lanes 2-3, PCR products from uninjected cells using the primer combinations P9/P7 (lane 2) and P9/P548 (lane 3). Lanes 4-5, PCR products from cells injected with pCE19 using the primer combinations P9/P7 (lane 4) and P9/P548 (lane 5). Lanes 6-7, PCR products from cells injected with pCE20 using the primer combinations P9/P7 (lane 6) and P9/P548 (lane 7). (b) The gel was hybridized with a DIG-labeled probe of the polylinker inserted into the 1.3 kb macronuclear precursor sequence. While in the case of pCE19 ((a) lane 5) no PCR product using the primer combination P9/P548 is visible in the ethidium bromide stained gel, a clear signal is observed after hybridization ((b) lane 5).

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