Figure 1.

Outline of the TEV-Cleavage System

(A) Schematic of the cohesin complex containing TEV-cleavable Rad21 (green), SMC1 (red), SMC3 (blue), and Scc3/SA (yellow). Cleavage of Rad21 by separase occurs in the flexible linker region. Arrowheads indicate the sites of insertion of TEV-recognition sequences (numbers refer to amino acid positions).

(B) Outline of the TEV-cleavage system showing two alternative methods to express TEV in vivo in flies. (a) UAS-TEV is controlled by the UAS/GAL4 system, enabling TEV expression by specific Gal4 driver lines. (b) TEV directly fused to the heat-shock promotor allows for its ubiquitous induction in a time-specific manner. (c) Once expressed, catalytically active TEV protease cleaves Rad21^TEV.

(C) Representation of the genomic region of the Rad21 locus. The Rad21 gene (CG17436) resides in the centric heterochromatin of chromosome 3L. The exon-intron structure of the Rad21 mRNA is shown in bold. EST-based transcript predictions of neighboring genes are depicted in lighter gray. The EP element GE50159 4 kb upstream of the transcriptional start of Rad21 is represented by a red triangle. The four independently generated imprecise excision mutants of Rad21 lack the chromosomal intervals indicated by solid, red lines. The Rad21 locus is missing in the γ-ray-induced deficiency Def 2-66 (dashed line). The scale bar is 10 kb.

(D) Pupal protein extracts were prepared before (t = −0.75 hr) and at different time points after a 45 min heat shock at 37°C (red arrow). Western blot analysis with antibodies against endogenous Rad21 (left panel) or myc (right panel) shows full-length Rad21^TEV (arrow) and the C-terminal TEV-cleavage product (arrowhead) as well as gRad21 (asterisk). V5-tagged TEV protease is detected by probing with v5 antibodies (open circle). Actin was used as a loading control. A molecular weight marker (in kDa) is shown on the left.