Figure 3

Disruption of the rasGEFM gene. (A) Southern blot of genomic DNA from parental strain (AX2) and rasGEFM null mutant (HSB61). Genomic DNA was digested with Eco RI, separated in 0.8% agarose gel, blotted onto nylon membrane and probed with probe a (rasGEFM N-terminal fragment corresponding to bp 0–617 of the cDNA clone), probe b (corresponding to bp 760–1518 of the rasGEFM cDNA clone) and probe c (bsr cassette). Three different probes were used to characterize the genomic locus of the mutant and the parental strain. In the rasGEFM null strain the central part of the gene (recognized by probe b), was replaced by the blasticidin cassette (recognized by probe c), which has the same size of the replaced fragment. Because of that, the size of the locus remains unchanged, but the central part is recognized specifically by bsr (probe c) or probe b in HSB61 or AX2, respectively. Both AX2 and HSB61 genomic loci are recognized by the probe a. (B) Northern blot of total RNA extracted from HSB61 cells at the indicated time points of growth and development. The membrane was hybridized to the rasGEFM and to the actin gene.