Generation of Deletion Constructs of DAZ and DAZL1
Deletion constructs of DAZL1 and DAZ were generated from cDNA clones DAZL1-f2 and DAZ-e11, respectively. Deletions were created either by using existing restriction sites within the coding regions or by polymerase chain reaction amplification of two regions followed by ligation. Correct joining at the junctions was verified by DNA sequencing. DAZL1-f2 encodes a protein of 295 residues, with the RRM domain spanning residues 32 to 115 and the DAZ repeat spanning 167 to 190. Deletion constructs DAZL-R1, DAZL-Z1, DAZL-C1, and DAZL-RZ1 encode proteins lacking residues 57-110, 169-192, 203295, and 57-110 plus 169-192, respectively. DAZ-e11 encodes a protein of 414 amino acid residues with 9 DAZ repeats. The RRM domain spans residues 32 to 115, and the DAZ repeat region spans 167 to 382. Deletion constructs DAZ-R1, DAZ-R2, DAZ-Z1, DAZ-Z2, and DAZ-RZ1 encode proteins lacking residues 57-110, 39-110, 206-373, 155-382, and 39-110 plus 206-373, respectively. Trx-RRM and Trx-DAZ were constructed by cloning restriction fragments of DAZ cDNA that contained the RRM domain and the DAZ repeats, respectively, in frame into the pET-32 vectors (Novagen). They encode fusion proteins between thioredoxin and DAZ fragments.
Detection of DAZL1 in Mouse Tissue Extracts
Tissues were removed from 2- to 3-mo-old mice immediately after they were killed. About 0.2 g of tissues were homogenized in 1 ml of an extraction buffer (EB) containing 20 mM Tris pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.3% Igepal CA-630 (Sigma), 40 U/ml ribonuclease inhibitor, 1 mM PMSF, and 1 ^g/ml each of aprotinin and leupeptin, in a Wheaton Potter-Elvehjem tissue grinder. Homogenates were centrifuged at 1000 X g for 10 min to remove cell debris and nuclei. The supernatants were centrifuged again at 10 000 X g for 10 min to generate a pellet containing largely mitochondria, and a postmito-chondrial supernatant (PMS). The pellet was resuspended in EB to a final volume equal to that of PMS. Analysis of aliquots on 10% SDS-PAGE gels was followed by Western blotting with anti-DAZL1 antibody.