Fig. 3. Analyses of ns alleles. (A) A hybridization probe (probe 1; Materials and methods) derived from a PRS-homologous, maize genomic clone identifies two distinct DraI restriction fragments linked to the ns mutant phenotype in F2 (ns-RxB73) segregating progeny. Note that no internal DraI restriction sites are present within the sequence of probe 1 obtained from either ns mutant or B73 individuals. (B) Composite gene map of the ns duplicate genes, each of which comprises two exons and a single intron. Exons are boxed, the positions of the homeodomain (HD), the glutamine-rich region (Q), the histidine-rich region (H) and the PLK domain are indicated. Solid lines indicate introns and untranslated regions. The position of the extra G in ns2-R and of the CACTA element insertion in ns1-R are indicated above the drawing. The dashed line below the drawing indicates regions of the ns2 locus that are deleted in the ns2-Mu1 allele. (C) Newly identified ns1-Mu^* alleles are deletions. Active mutator lines Ns1/Ns1; ns2-R/ns2-R (ns1-Mu6/7 Mu parent) were pollinated with ns1-R/ns1-R; ns2-R/ns2-R (ns-R parent) pollen and analyzed for phenotypic progeny in the M1 generation. Southern analysis of ns mutant progeny (shown are ns1-Mu1 or ns1-Mu3) showed lack of the ns1 wild-type (6 kb) band but presence of the ns1-R (4.5 kb) fragment when hybridized to probe 2 (Materials and methods), whereas wild-type siblings (ns1-Mu1/3 sibs) always contained both bands (6 kb + 4.5 kb). Note that probe 2 hybridizes more weakly to ns2 because it includes 5' UTR sequence specific to the ns1 locus. (D) Plants homozygous for the ns2-^*Mu1 mutant allele exhibit no hybridizing restriction fragment corresponding to the ns2 locus, whereas non-mutant siblings exhibit a 3.5 kb band linked to ns2. (E) NS protein does not accumulate in young ears obtained from ns1-R, ns2-R mutant plants. Polyclonal antibodies raised against an oligopeptide that is conserved in the predicted NS1 and NS2 proteins recognize a protein (arrow) in homozygous non-mutant ears, as well as in non-mutant ears from plants that contain a single non-mutant allele of Ns1 but are homozygous for the ns2-R mutant allele. This 29 kDa band is absent in ears from ns mutant plants, and corresponds to the predicted molecular weight of the NS proteins. Note that the NS polyclonal antibody is not specific for NS proteins.