Fig. 1.

Ustilago maydis Lis1 protein and effect of its depletion on growth. A. Domain organization of the Ustilago maydis Lis1 protein. Lis H =Lis1 homology domain (amino acid residues 20–51); cc = coiled coil domain (aa residues 69–95); WD = tryptophan-aspartic acid repeats. Analysis was carried out with Expasy Prosite (Hulo et al. 2007, http://www.expasy.ch/prosite/) and Coils (Lupas et al. 1991, http://www.ch.embnet.org/software/COILS_form.html). B. Gene replacement of the wild type lis1 allele with the Pcrg1∷lis1 allele by homologous recombination. The targeting fragment was generated in vitro. X = XhoI. The XhoI fragment in the wild type allele is 3000 bp and in the Pcrg1∷lis1 allele 5544 bp because the hygB gene introduces a XhoI site, as indicated. C. Southern hybridization analysis of FB1 (a1 b1) transformants. a = FB1, b = FB2, c–j = independent transformants. Gene replacement occurred in strains d, e, f and g, as evidenced by the presence of the diagnostic 5544 bp band (arrow) and the absence of the wild type band (3000 bp, arrowhead). M = molecular weight markers consisting of a mixture of DIG-labeled MW marker II and VI (Roche). The top panel was probed with a 550 bp probe from the lis1 ORF region, and the bottom panel is the same membrane reprobed with a 872 bp probe from the hygB gene. D. Southern hybridization analysis of SG200 (a1 mfa2 bW1 bE2) transformants. a = FB1, b = SG200, c–o = independent transformants. Gene replacement occurred in all transformant strains analyzed (c–o) as evidenced by the presence of the diagnostic 5544 bp band (arrow) and the absence of the wild type band (arrowhead). M = molecular weight markers as in Panel C. Top and bottom panels were probed as indicated for Panel C. E. Growth of wild type and Pcrg1∷lis1 strains on YEP glucose. Strains were grown as indicated, serially diluted, spotted on YEP glucose medium and incubated at 28 C for 3 d. 1 = FB1 (a1 b1); 2 = FB1 Pcrg1∷lis1; 3 = SG200 (a1 mfa2 bW1 bE2); 4 = SG200 Pcrg1∷lis1.