Agarose

pancreatic ribonuclease (solubilizing peptide) and with two flanking Hisx6 tags (Fig. 1A). Purification of the recombinant proteins was achieved by lysing the transformed bacteria in 20 mM MOPS pH 7.5, followed by single step affinity purification on Ni-agarose and subsequent size exclusion chromatography to eliminate the imidazole used to elute the recombinant proteins. SDS-PAGE analysis (Fig. 1B, first panel) showed major bands at the MW of 34 kDa (rGRA2), 27 kDa (rGRA2 (NT-CT)) and 43 kDa (rGRA6), respectively

(Pharmacia) for primer extension analysis and by the chemical method (Sambrook et al., 1989) for DNase I footprinting. Pfu DNA polymerase (Stratagene) was used for preparative PCR as recommended by the manufacturer. DNA fragments were purified from agarose gels using the gel extraction kit (Qiagen). Plasmid constructions Reporter fusions of promoter fragments to a promoterless lacZ gene were constructed using the vector pDG1661 (Guérout-Fleury et al., 1996). The promoter fragments to be tested were

moléculaire, permettent de séparer les acides nucléiques en fonction de leur taille: L'électrophorèse horizontale sur gel d'agarose permet de séparer les fragments d'ADN de 300 à 10 000 paires de bases en fonction de la concentration du gel en agarose L'électrophorèse verticale sur gel de polyacrilamide permet de séparer les fragments d'ADN dont les longueurs vont de 1 à 1 000 nucléotides en fonction de la longueur du gel et de la tension appliquée (de 1 000 à 2000 volts / cm). L’électrophorèse

explain why, so far, no fungal integrin b-related molecules have been identified. METHODS Cell culture and mutagenesis. Cells were cultured at 21 1C in HL5 medium (Cornillon et al, 1998). To initiate multicellular development, cells were plated on 1% agarose Petri dishes in nutrient-free medium (5 mM Na2HPO4, 5 mM KH2PO4, 1 mM CaCl2, 2 mM MgCl2, pH 6.5; Sussman, 1987). The sibA mutant was isolated following a procedure described previously (Cornillon et al, 2000). The plasmid rescued from the original