<Original article>The Genomic Organization, Alternative Splicing, and Promotor Assay of the Mouse Ankhzn Gene

We have previously established a GT3-12 mouse line in which a novel gene was trapped by a promoterless gene trap (GT) vector. The gene was designated as Ankhzn because of the presence of 17 ankyrin-repeats and a zinc-finger domain FYVE. In this study, we characterize the genomic organization of the mouse Ankhzn gene by analyzing two overlapping P1 (bacteriophage P1 cloning system) clones and a single bacterial artificial chromosome (BAC) clone. The Ankhzn gene spans more than 95 kb and comprises 25 exons, where the splice site conforms to the GT-AG rule except for the GC splice donor site instead of GT of intron 4. This GC splice donor site and several junctions are conserved between the mouse Ankhzn and the human ANKHZN. Furthermore, results of RT-PCR revealed that the 49 bp segment at the 3' end of exon 9 is alternatively spliced to generate two splice variants. One splice variant is a long form that has a calculated molecular mass of 130 kDa. The other is a short form that has a molecular mass of 50 kDa resulting from a frameshift leading to premature termination within exon 10. Results of RT-PCR analysis showed that the Ankhzn long form was expressed much more strongly than the short form in all tissues and developmental stages examined. The Ankhzn gene was identified as a single-copy gene by Southern blot analysis. Examination of the T31 Mouse Radiation Hybrid Database RH Chr 11 Public Map Data at the Jackson Laboratory showed its localization to mouse chromosome 11 which has high synteny to human chromosome 17. A promoter assay with a luciferase reporter gene revealed that an approximately 200 bp upstream region to the transcription start site is essential for the transcription of Ankhzn.