To isolate the murine
nyx gene, a cDNA clone (GenBank accession no. AI861796; http://www.ncbi.nlm.nih.gov/Genbank/; GenBank is provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD), containing a fragment of cDNA from the human
NYX gene was obtained from the Image consortium (http://www.info@image.llnl.gov/; provided in the public domain and hosted by The Lawrence Livermore National Laboratory, Livermore, CA). The cDNA insert was isolated, radiolabeled by the random-primed oligo-labeling method (Amersham Pharmacia Biotech, Piscataway, NJ), and hybridized to BAC clones encompassing the
nob critical region. For two positively hybridizing BACs,
EcoRI and
HindIII shotgun libraries were constructed and screened. A clone containing a 5.9-kb
EcoRI fragment was identified from two different BACs and found through sequencing to contain exon 3 of the
nyx gene. The remainder of the murine
nyx gene was isolated by cloning and sequencing fragments that overlapped this clone. DNA fragments were sequenced using fluorescent cycle sequencing and analyzed on a DNA sequencer (model CEQ2000XL; Beckman-Coulter, Fullerton, CA). The sequence of the murine
nyx gene was obtained by a combination of end sequencing of
EcoRI and
HindIII subclones of one BAC clone (402g19), primer walking, and import of sequences from the Ensembl trace database (http://www.ensembl.org/; provided in the public domain by the European Molecular Biology Lab [EMBL], Heidelberg, Germany, and the Sanger Centre, Hinxton, UK).
14 The predicted coding region was determined from expressed sequence tag EST and genomic sequence. The several Internet-accessible resources that were used to analyze the
nyx protein sequence will be indicated later.