THE E-SUBUNIT OF VACUOLAR H-ATPASE LOCALIZES CLOSE TO THE CENTROMERE ON HUMAN-CHROMOSOME 22()

As part of a general effort to identify new genes mapping to disease-a ssociated regions of human chromosome 22, we have isolated heterogeneo us nuclear RNA from somatic cell hybrids selected for their chromosome 22 content. Inter-Alu PCR amplification yielded a series of human DNA fragments which all detected evolutionarily-conserved sequences. The centromere-most gene fragment candidate, XEN61, was shown to tie centr omeric to the chromosome 22 breakpoint in the X/22-33-11TG somatic cel l hybrid. This region, which is still devoid of characterized genes, o verlaps with the critical region for the cat eye syndrome (CES), a dev elopmental disorder associated with chromosomal duplication within 22p ter-q11.2. Gene dosage analysis performed on DNA from six CES patients consistently revealed the presence of four copies of XEN61. A fetal b rain cDNA clone, 61EW, was identified with XEN61 and entirely sequence d. The deduced protein is the E subunit of vacuolar H+-ATPase. This 31 KDa component of a proton pump is essential in eukaryotic cells as it both controls acidification of the vacuolar system and provides it wi th its main protonmotive force. RT-PCR experiments using oligonucleoti des designed from the 61EW cDNA sequence indicated that the correspond ing messenger is widely transcribed.