Fig. 4. Positional cloning of npo. (A) Integrated genetic/physical map depicting chromosome walk across the npo locus, initiated at microsatellite marker z8532. Two overlapping BACs covered the npo interval, and these were subjected to complete sequencing, resulting in a 129 kb contig. Within this interval, we identified four genes and new microsatellite markers. These were used to genotype the flanking recombinants and to exclude the two outer genes. Thus only epha2 and a hypothetical RRM protein encoding gene fine-mapped within the genetic locus. We isolated full-length cDNAs corresponding to both genes from homozygous-npo mutant and wild-type embryos, and six independent clones from each were completely sequenced in both directions. No mutations were found in the epha2 coding sequence, whereas a T to A (encoding a Y to stop) mutation was identified in the RRM-encoding gene, which we designated npo. (B) Sequence tracing of one of 12 independent clones derived from PCR of genomic DNA from mutant and wild-type embryos, showing TAT (Y) to TAA (stop) mutation. (C) cDNA from wild-type and mutant embryos subjected to in vitro transcription/translation produces a truncation of mutant Npo, in agreement with the conceptual translation. The doublet most likely arises from an alternate translation start site.