Gene Structure for Adenosine Kinase in Chinese Hamster and Human: High-Frequency Mutants of CHO Cells Involve Deletions of Several Introns and Exons

Abstract

The structure for the adenosine kinase (AK) gene has been determined from Chinese hamster (CH) and human cells. The AK gene in CH is comprised of 11 exons ranging in length from 36 to 765 nt, with the majority<100 nt. The exact lengths of the intervening introns have not been determined, but most of them are indicated to be very large (>15 kb). A 6.6-kb fragment from human cells was also sequenced, and it contained only a single exon corresponding to exon 10 in CH. The BLAST searches of the subsequently released draft human genome sequence have revealed that the AK gene structure in human is identical to that in CH. In the human genome, the AK exons are distributed over four genomic clones totaling 752 kb, providing direct evidence that the AK gene in mammalian species is unusually large. In contrast to CH and human, the AK genes from several other eukaryotic organisms whose complete genomes are now known are quite small (between 1.2 and 2.5 kb) and either contain no introns (Saccharomyces cerevisiae and Schizosaccharomyces pombe) or various numbers of introns (Drosophila melanogaster [2], Caenorhabditis elegans [4], Arabidopsis thaliana [10]). Some of the intron-exon junctions in these species are in the same positions as in mammals. The AK gene in CH and human, as well as mouse, is linked upstream in a head-to-head fashion with the gene for the clathrin adaptor mu3 protein (or beta3A subunit of the AP-3 protein complex), which is affected in type 2 Hermansky-Pudlak syndrome. These two genes are separated by <200 nt, and it is possible that they have a common or overlapping promoter(s). We have also determined the nature of the genetic alterations in two of the class A AK^- mutants of CHO cells, which are obtained at a very high spontaneous frequency (10^-3-10^-4) in this cell line. Both mutants contained large deletions within the AK gene and greatly shortened AK transcripts. The cloning and sequencing of the transcripts from these mutants showed that the deletion in one of them led to the loss of exons 5 through 8, whereas in the other, all exons from 2 through 8 are deleted. The endpoints of these deletions lie in the large introns within the AK gene.