Figure 6

Characterization of strains where sigN genes have been over-expressed, repressed by the use of interfering RNA or deleted. A. Total protein extracts were obtained from WT (AX4) and three strains (2, 4, 11) transfected with a plasmid vector for sigN4 over-expression. Group 1 antibodies were used for Western-Blot. The migration of the 10 and 15 kDa protein markers is shown on the left. B. RNA was obtained from WT and two different RNAi clones that expressed an interfering RNA based on exon 2 of sigN3 (1 and 6 clones) after 14–18 hours of development on Nitrocellulose filters. Arrows indicate the position of the endogenous mRNA (sigN mRNA) and of the RNAi transcribed from the vector (RNAi construct). Ethidium bromide staining of the gel is shown in the lower panel. C. RNA from WT (AX4) and Group 1 KO strains, harvested at the indicated hours of development (16–18 h) was analyzed by RT-PCR for expression of sigN Group 1 genes. The control band obtained by amplification of a region of the large mitochondrial ribosomal RNA (control: 320 bp long) and that corresponding to sigN Group 1 mRNA (sigN:240 bp) are indicated by arrows at the right. D. RNA was obtained from WT (AX4) and two different Group 2 KO strains (KO12, KO20) after 16 hours of development. Expression of sigN Group 2 genes was analyzed by RT-PCR. The migration of the control band (control: 320 bp) and that corresponding to sigN Group 2 mRNA (sigN: 240 bp) is indicated at the right. E. DNA from WT (AX4) and a Group 1 and Group 2 Double KO strain (Double KO) was analyzed by PCR for the presence of for sigN Group 1 genes. Migration of the amplified fragments for a control gene (control: 600 bp) and sigN Group 1 genes (410 bp) is indicated at the right.