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.     

Overlapping two genes in human DNA: a salivary amylase gene overlaps with a gamma-actin pseudogene that carries an integrated human endogenous retroviral DNA

The human salivary amylase gene (amy1), consisting of eleven exons, is expressed in the salivary gland and in some amylase-producing tumors. Its uppermost exon and the following intron, along with the 5'-flanking region of this gene, are shown to be superimposed with a gamma-actin pseudogene sequence, a portion of which is transcribed into salivary amylase mRNA and another portion of which serves as a promoter for the amy1 gene. In the further upstream region, the gamma-actin pseudogene sequence is interrupted by a human endogenous retroviral nucleotide sequence.     

Mapping the human amylase gene cluster on the proximal short arm of chromosome 1 using a highly informative (CA)n repeat.

The human amylase gene cluster includes a (CA)n repeat sequence immediately upstream of the gamma-actin pseudogene associated with the AMY2B gene. Analysis of this (CA)n repeat by PCR amplification of genomic DNA from the 40 families of the Centre d'Etude du Polymorphisme Humain (CEPH) reference panel revealed extensive polymorphism. A total of six alleles with (CA)n lengths of 16-21 repeats were found. The average heterozygosity for this polymorphism was 0.70. Multipoint linkage analysis showed that the amylase gene cluster is located distal to the nerve growth factor beta-subunit gene (NGFB) and is within 1 cM of the anonymous locus D1S10. The amylase (CA)n repeat provides a convenient marker for both the physical and the genetic maps of human chromosome 1p.     

Evolution of the human alpha-amylase multigene family through unequal, homologous, and inter- and intrachromosomal crossovers

Human amylase haplotypes differ from each other by different numbers of a long direct repeat unit of approximately 100 kb, encompassing two complete salivary amylase genes and one amylase pseudogene lacking the first three exons. The two salivary genes are part of a 75-kb-long inverted repeat. Two short sequences, hybridizing with a probe containing exons 1-3, were found in the central part of the inverted repeat. Sequencing showed that these fragments, designated r, contain exon 3 sequences. We present evidence that these r-fragments and the pseudogene most likely are remnants of the same ancestral pancreatic gene. We determined the orientation of the exon 3 sequences present in the r-fragment and show that an inversion can explain their origination. Hybridization studies, using random fragments from the intergenic region of the AMY gene cluster as probes, enabled us to detect more extended homologous regions in this cluster than were found previously on the basis of restriction maps only. Together, these results allow us to present a model for the evolution of the human amylase multigene family by a number of consecutive events involving inter- and intrachromosomal crossovers.     

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.     

The human alpha-amylase multigene family consists of haplotypes with variable numbers of genes

Polymorphic amylase protein patterns have suggested the presence in the human genome of various haplotypes encoding these allozymes. To investigate the genomic organization of the human alpha-amylase genes, we isolated the pertinent genes from a cosmid library constructed of DNA from an individual expressing three different salivary amylase allozymes. From the restriction maps of the overlapping cosmids and a comparison of these maps with the restriction enzyme patterns of DNA from the donor and family members, we were able to identify two haplotypes consisting of very different numbers of salivary amylase genes. The short haplotype contains two pancreatic genes (AMY2A and AMY2B) and one salivary amylase gene (AMY1C), arranged in the order 2B-2A-1C, encompassing a total length of approximately 100 kb. The long haplotype spans about 300 kb and contains six additional genes arranged in two repeats, each one consisting of two salivary amylase genes (AMY1A and AMY1B) and a pseudogene lacking the first three exons (AMYP1). The order of the amylase genes within the repeat is 1A-1B-P1. All genes are in a head-to-tail orientation except AMY1B, which has the reverse orientation with respect to the other genes. Analysis of somatic cell hybrids confirmed the presence of these short and long haplotypes. Furthermore, we present evidence for the existence of additional haplotypes in the human population and propose a general model for the evolution of the human alpha-amylase multigene family. A general designation 2B-2A-(1A-1B-P)n-1C can describe these haplotypes, n being 0 and 2 for the short and the long haplotypes presented in this paper, respectively.     

Interpretation of polymorphic DNA patterns in the human alpha-amylase multigene family.

Previous molecular studies have clearly shown that the human amylase locus has a very complicated structure. Multiple salivary and pancreatic amylase genes are present on haplotypes with variable numbers of genes. To study the population heterogeneity, human genomic DNA from family members and random individuals was digested with a number of different restriction enzymes and hybridized with probes representing various parts of the human pancreatic amylase cDNA. The complex patterns obtained were, in most cases, compatible with predictions from the restriction enzyme maps of cloned human amylase genes. With some enzymes deviations from the predicted intensities of the bands associated with the pancreatic amylase gene AMY2A were observed. These findings can be explained by unequal homologous crossovers between AMY2A and AMY1A, resulting in haplotypes with one gene less or one gene more than the haplotypes described thus far. Moreover, a very complicated TaqI polymorphism was found that can be explained by homologous crossovers between different salivary amylase genes. Because some salivary amylase genes have an inverted orientation with respect to the others, these data provide evidence for the occurrence of intrachromosomal, homologous crossovers, as proposed by us previously (P. C. Groot et al., 1990, Genomics 8: 97-105).     

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.     

Evidence for duplication of the human salivary amylase gene

Summary Isoelectric focusing of human parotid saliva reveals different -amylase patterns reflecting qualitative and quantitative variations. A puzzling pattern, which shows three different amylase gene products, was found in four individuals. Based on this observation a model is presented in which the salivary amylase gene is duplicated. Family studies show that the AMY1 ^* A2 gene forms a haplotype with the normal gene, AMY1 ^* A1, whereas the AMY1 ^* A3 gene still exists in a single form. The absence of homozygote 2-2 in offspring of 1-2x1-2 marriages and in population material, and the fact that the variant protein makes up about only 20–30% of the total amylase protein in heterozygotes can be considered as additional evidence supporting the hypothesis. The possibility that cis-acting regulatory variants are involved in the patterns with quantitative variation is discussed.     

Cloning and characterization of a third type of human alpha-amylase gene, AMY2B

We have previously reported concerning the existence of a third type of human alpha-amylase gene, AMY3 [Emi et al., Gene 62 (1988) 229-235; Tomita et al., Gene 76 (1989) 11-18], which is expressed in a lung carcinoid tissue, and differs in nucleotide sequence from the two previously characterized human alpha-amylase genes coding for salivary and pancreatic isozymes, termed AMY1 and AMY2, respectively. Here, we rename this gene AMY2B to coincide with the designation by Gumucio et al. [Mol. Cell Biol. 8 (1988) 1197-1205] and describe its genetic properties as revealed by sequencing studies. It consists of ten major exons whose sequences are highly homologous to those of AMY1 and AMY2. Not only the exons, but also most of the introns seem to be highly conserved, as judged from physical mapping data. The AMY2B gene identified from mRNA in a lung carcinoid tissue has at least two additional untranslated exons in its 5' region; hence the promoter lies far upstream relative to the other two AMY genes.     

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.     

Electrophoretic behavior of amylase in mouse: the parotid gland is major source of serum amylase

Amylase activity in the saliva, salivary glands, serum, liver (perfused and non perfused) and pancreas was assayed and isoamylases were separated by electrophoresis in these organs using C57BR/cdJ and M. m. molossinus (Kor) mice. Amylase isozymes in the saliva, parotid gland, serum and liver were identical in both strains, respectively. Amylase activity in the liver was lower than that in the serum and liver amylase disappeared almost by perfusion. Major serum amylase was released from the parotid gland in intact animals.