Structure And Evolution Of The Amylase Multigene Family (cosmids, Human Amylase, Chromosome Walking, Mouse).

Abstract

The mammalian amylase multigene family is composed of several closely linked and structurally homologous genes which are regulated in a highly tissue-specific manner. To study the mechanisms involved in their differential regulation, we have isolated representative of all of the amylase genes of the mouse and human genomes. The use of cosmid vectors has enabled the isolation of intact genes as well as several kilobases of flanking DNA which could contain important regulatory sequences. We found that, in mouse strain YBR/Ki, the amylase multigene family consists of two nearly identical copies of the salivary amylase gene, Amy-1, a single copy of each of two divergently regulated pancreatic amylase genes, Amy-2.1 and Amy-2.2, and a pancreatic amylase pseudogene. When compared to most other inbred mouse strains, this represents an expansion of the Amy-1 locus and a contraction of the Amy-2 locus. This accounts for the unusual amylase phenotype of the YBR/Ki strain, which produces twice as much salivary amylase and half as much pancreatic amylase as other strains. To study the mechanisms involved in expansion and contraction of this gene family, the organization of the amylase genes on mouse chromosome 3 was determined by chromosome walking. These studies revealed the tandem arrangement of three genes, in the same 5' to 3' orientation: Amy-1 - 22 kb - Amy-2.1 - 10 kb - pseudogene. Parallel studies in the human genome revealed more about the evolutionary history of this multigene family. Six different types of human amylase genes were isolated. We found that the human Amy-1 locus contains three closely related gene copies. Sequence analysis showed that, as in the mouse, an intron interrupts the 5' non-translated portion of the human Amy-1 transcript. The nearly identical position of this intron in the two species demonstrates that the Amy-1 genes of human and mouse are derived from a common ancestor, and did not arise by independent gene duplication. In both species, clear evidence for gene conversion was also found. Since DNase I hypersensitive sites are often associated with important regulatory sequences, we studied the distribution of these sites within the well-characterized mouse amylase cluster. Several such sites were identified which were directly correlated with expression of the amylase genes. Exonuclease protection was used to assay nuclear extracts for the presence of proteins which could bind to specific sequences in cloned DNA.