Figure 4

A) Deletion in C. elegans nfi-1 gene. The relative locations of confirmed exons (boxes) are shown. Square brackets indicate the location of the NFI-1 DNA-binding domain and the region deleted in nfi-1 mutant. The arrows indicate the position of the Tc1 insertion in an intron of the nfi-1 gene in strain NL747 pk240 and locations of PCR primers used in the screening for the deletion. B) Single-worm PCR reactions on N2 worm and nfi-1 homozygous mutant isolated by sib-selection. The arrows indicate a 1007 bp and 2969 bp PCR products corresponding to the nfi-1 mutant (qa524) and wild type alleles respectively. Lane 2 is 1 kb DNA ladder. C) RT-PCR on N2 worms and nfi-1 homozygous mutants. The arrows indicate a 480 bp and 320 bp RT-PCR products amplified using total RNA obtained from N2 worms (lane 1). nfi-1 mutants show loss of nfi-1 transcripts (lane 2). Lane (3) is 100 bp DNA ladder. D) Loss of NFI DNA-binding activity in extracts of nfi-1 mutants. Nuclear extracts of a mixed population of N2 worms and nfi-1 mutants were prepared and used in a gel mobility shift assay with an oligonucleotide (2.6) that contains an NFI-binding site (lanes 3, 5) or the same oligo with a single point mutation that abolishes NFI binding (C2) (lane 4, 6). See Fig. 2A for sequences of oligonucleotides. Extract of nfi-1 mutants show loss of NFI DNA-binding activity (lanes 5, 6). Lanes 1 & 2, no extract.