Interstrain Variation in Amylase Gene Copy Number and mRNA Abundance in Three Mouse Tissues

Amylase expression in strain YBR differs in several respects from the standard mouse phenotype. The synthesis of salivary amylase is elevated twofold in YBR mice and the synthesis of pancreatic amylase is reduced to one-half the normal rate. We have compared the concentrations of amylase mRNA in the parotid, liver and pancreas of YBR mice with those in strains A/J and C3H. We observed differences in amylase mRNA abundance which can account for the levels of amylase protein synthesis in the parotid and pancreas of these strains. Unexpectedly, the concentration of amylase mRNA in the liver of YBR mice was also higher than in the other strains. Since liver amylase is transcribed from the same gene as parotid amylase, duplication of the Amy-1 locus could account for the elevated mRNA concentration in both tissues. Quantitative analysis of genomic DNA by Southern blotting provided direct evidence for duplication of Amy-1 in strain YBR.     

Tissue-specific and insulin-dependent expression of a pancreatic amylase gene in transgenic mice.

The regulatory properties of mouse pancreatic amylase genes include exclusive expression in the acinar cells of the pancreas and dependence on insulin and glucocorticoids for maximal expression. We have characterized a murine pancreatic amylase gene, Amy-2.2y, whose promoter sequence is 30% divergent from those of previously sequenced amylase genes. To localize sequences required for tissue-specific and hormone-dependent activation, we established two lines of transgenic mice. The first line contained a single copy of the complete Amy-2.2y gene as well as 9 kilobases of 5'-flanking sequence and 5 kilobases of 3'-flanking sequence. The second line carried a minigene which included 208 base pairs of 5'-flanking sequence and 300 base pairs of 3'-flanking sequence. In both lines the transgene was expressed at high levels exclusively in the pancreas. Both constructs were dependent on insulin and induced by dexamethasone. Thus, the transferred genes contained the sequences required for tissue-specific and hormonally regulated expression.     

Two Distinct Pancreatic Amylase Genes Are Active in Ybr Mice

The genetic determinants of pancreatic amylase expression in YBR mice differ in two respects from those of other inbred strains. First, there are two nonallelic amylase isozymes present in YBR pancreas, while most mouse strains express a single pancreatic amylase protein. In addition, the in vivo rate of total pancreatic amylase synthesis is 50% of that in other strains. Both these traits are determined by genetic sites in the region of the Amy-2 locus on mouse chromosome 3. To determine the molecular basis for the presence of two isozymes in this strain, we have compared portions of their amino acid sequences. Two differences between isozymes A₁ and B₁ were identified among the 77 residues compared. This result demonstrates that two distinct amylase genes are expressed in YBR pancreas.     

The structure of two distinct pancreatic amylase genes in mouse strain YBR

The amylase complex on mouse chromosome 3 encodes both salivary and pancreatic amylase. It appears that one active gene is present for salivary amylase, whereas pancreatic amylase in some strains is coded by at least 4, and perhaps by more than 10, genes. Strain YBR is different from other strains in that it produces twice as much salivary amylase. Pancreatic amylase in YBR is present as two different protein forms, A and B, the sum of which amounts to only one-third of that in, for instance, strain A/J. YBR chromosomal DNA was cloned in phage , followed by restriction and heteroduplex analysis of recombinant phages carrying amylase genes. Among 32 phage isolates, 5 carried parts of the salivary amylase sequence. The remaining phage isolates contained pancreatic amylase-like sequences and represented three nonoverlapping genomic regions, i.e., one of 34 kb containing a complete gene, PAN-II; another of 41 kb with a complete but different gene, PAN-I, plus a truncated gene, PAN-1; and finally, one of 23 kb with another truncated gene, PAN-2. Parts of the amino acid sequence of A and B have previously been determined, and we report here the sequencing of a 4-kb DNA fragment from Pan-II which establishes that this gene codes for B.This work was supported by the Danish Natural Science Research Council.     

Binding of a pancreatic nuclear protein is correlated with amylase enhancer activity.

The mouse amylase gene Amy-2.2 is expressed at high levels specifically in the acinar cells of the pancreas. The region between -172 and -110 of this gene includes sequence elements common to pancreas-specific genes. Nuclear proteins with specific affinity for this region were partially purified from rat pancreas. The consensus element of another pancreas-specific gene, elastase 1, competes for protein binding to the amylase sequences. Binding was localized by DNase I protection to the sequence -156 to -122. Site-directed mutagenesis of this sequence resulted in concomitant loss of protein binding and enhancer activity. Photo-affinity labelling of pancreatic nuclear extracts identified one predominant binding protein with a molecular weight of approximately 75 kDa. The data indicate that binding of this nuclear protein is essential for the enhancer activity of this pancreas-specific element.     

Multiple structural genes for mouse amylase

Salivary and pancreatic amylases from the mouse show both structural and quantitative genetic variation encoded within a gene complex on chromosome 3. Two fundamental questions prompted by this variation are whether salivary and pancreatic amylases are derived from different structural genes and whether multiple structural genes are causing the quantitative variation observed in each of the two amylases. These questions were approached by comparing the amylase protein from 12 congenic lines carrying amylase gene complexes derived from different origins. The amylases were purified by affinity chromatography employing the inhibitor cyclohepta-amylose and characterized in terms of amino acid composition, specific activity, molecular weight, and heat stability. They were analyzed by native electrophoresis in polyacrylamide gels and by peptide mapping employing both cyanogen bromide cleavage and restricted proteolysis in the presence of dodecylsulfate. By these techniques, many differences in the structure of pancreatic amylase that were not reflected in the salivary amylase were found among mouse strains. Likewise, a distinct salivary amylase variant was found. These results suggest that independent structural genes exist for the two amylases. Furthermore, by all criteria used, pancreatic amylase from single strains exhibits molecular heterogeneity, whereas heterogeneity was never found for salivary amylase. We conclude that at least four structural genes code for pancreatic amylase while only a single gene, different from any of the pancreatic genes, codes for salivary amylase.This work was supported by grants from the Danish Natural Science Research Council and a grant from the United States Public Health Service (Grant GM-19521). Part of the study was made during a 1-month visit of A. J. L. in Aarhus, which was supported by grants from NATO and the University of Aarhus.     

Simultaneous expression of salivary and pancreatic amylase genes in cultured mouse hepatoma cells.

The tissue-specific expression of two types of mouse amylase genes does not overlap in vivo; the Amy-1 locus is transcribed in the parotid gland and the liver, while expression of Amy-2 is limited to the pancreas. We identified a mouse hepatoma cell line, Hepa 1-6, in which both amylase genes can be simultaneously expressed. Amy-1 is constitutively active in these cells and is inducible by dexamethasone at the level of mRNA. We demonstrated that the liver-specific promoter of Amy-1 is utilized by the dexamethasone-treated hepatoma cells, and that glucocorticoid consensus sequences are present upstream of this promoter. Amy-2 is not detectable constitutively, but can be activated if the cells are cultured in serum-free medium containing dexamethasone. Expression of Amy-2 in a nonpancreatic cell type has not previously been observed. We speculate that induction of Amy-1 and activation of Amy-2 may involve different regulatory mechanisms. Hepa 1-6 cells provide an experimental system for molecular analysis of these events.     

On the mechanism of variation of pancreatic amylase levels in mouse strains

The relative levels of pancreatic amylase mRNA closely parallel the 3-fold variation of pancreatic amylase protein levels observed in several mouse strains studied. In contrast pancreatic elastase mRNA levels were similar in these strains. Each of the strains contained the same number of amylase-like genes (about 8). The selective variation in the pancreatic amylase gene expression could be due either to differences in the numbers of active amylase genes or to different rates of synthesis and/or degradation of the amylase mRNA from a constant number of genes.     

Retroviral and pseudogene insertion sites reveal the lineage of human salivary and pancreatic amylase genes from a single gene during primate evolution.

We have analyzed the junction regions of inserted elements within the human amylase gene complex. This complex contains five genes which are expressed at high levels either in the pancreas or in the parotid gland. The proximal 5'-flanking regions of these genes contain two inserted elements. A gamma-actin pseudogene is located at a position 200 base pairs upstream of the first coding exon. All of the amylase genes contain this insert. The subsequent insertion of an endogenous retrovirus interrupted the gamma-actin pseudogene within its 3'-untranslated region. Nucleotide sequence analysis of the inserted elements associated with each of the five human amylase genes has revealed a series of molecular events during the recent history of this gene family. The data indicate that the entire gene family was generated during primate evolution from one ancestral gene copy and that the retroviral insertion activated a cryptic promoter.     

Concerted evolution of human amylase genes.

Cosmid clones containing 250 kilobases of genomic DNA from the human amylase gene cluster have been isolated. These clones contain seven distinct amylase genes which appear to comprise the complete multigene family. By sequence comparison with the cDNAs, we have identified two pancreatic amylase genes and three salivary amylase genes. Two truncated pseudogenes were also recovered. Intergenic distances of 17 to 22 kilobases separate the amylase gene copies. Within the past 10 million years, duplications, gene conversions, and unequal crossover events have resulted in a very high level of sequence similarity among human amylase gene copies. To identify sequence elements involved in tissue-specific expression and hormonal regulation, the promoter regions of the human amylase genes were sequenced and compared with those of the corresponding mouse genes. The promoters of the human and mouse pancreatic amylase genes are highly homologous between nucleotide -160 and the cap site. Two sequence elements thought to influence pancreas-specific expression of the rodent genes are present in the human genes. In contrast, similarity in the 5' flanking sequences of the salivary amylase genes is limited to several short sequence elements whose positions and orientations differ in the two species. Some of these sequence elements are also associated with other parotid-specific genes and may be involved in their tissue-specific expression. A glucocorticoid response element and a general enhancer element are closely associated in several of the amylase promoters.     

Multiple non-allelic genes encoding pancreatic alpha-amylase of mouse are expressed in a strain-specific fashion.

The number of active Amy-2 genes has been estimated in strain CE/J mice which produce four distinct electrophoretic forms of alpha-amylase in their pancreas. cDNA cloning and DNA sequence analysis discloses five distinct mRNA sequences which differ by approximately 1% of their nucleotides. Two of these mRNAs specify the same protein. Changes in the nucleotide sequences result in amino acid replacements that alter the net charges of the deduced proteins. This has allowed a tentative assignment of individual mRNAs to isozymes detected by electrophoresis. Quantitative Southern blot hybridization using a DNA probe specific for the first exon of Amy-2 reveals the presence of greater than 10 Amy-2 related sequences per haploid CE/J genome. Models which could account for the mouse strain-specific differences with respect to the number of pancreatic alpha-amylase isozymes and their variable but genetically determined quantitative ratios are discussed.     

Genetic variation in mouse salivary amylase rate of synthesis

Heterozygotes from matings of the mouse strains YBR/Cv and C3H/As have about 3 times more YBR-amylase than C3H-amylase in the saliva. The determinant for this quantitative effect is located on linkage group XVI close to or within the structural gene for salivary amylase. The quantitative effect is the result of an increase in the rate of synthesis of YBR-amylase, and the determinant is cis acting. Studies of other mouse strains suggest that regulatory genetic elements may modulate salivary amylase production.This work was supported by the Danish Natural Science Research Council and a grant from the United States Public Health Service (Grant GM-19521).