The cell-specific elastase I enhancer comprises two domains.

Two separate domains within the 134-base-pair rat elastase I enhancer and a third domain at the enhancer-promoter boundary are required for selective expression in pancreatic acinar cells. The domains were detected by a series of 10-base-pair substitution mutations across the elastase I gene regulatory region from positions -200 to -61. The effect of each mutant on the pancreas-specific expression of a linked chloramphenicol acetyltransferase gene was assayed by transfection into pancreatic 266-6 acinar cells and control NIH/3T3 cells. The two enhancer domains are nonredundant, because mutations in either eliminated (greater than 100-fold reduction) expression in 266-6 cells. DNase I protection studies of the elastase I enhancer-promoter region with partially purified nuclear extracts from pancreatic tissue and 266-6 cells revealed nine discrete protected regions (footprints) on both DNA strands. One of three footprints that lie within the two functional domains of the enhancer contained a sequence, conserved among several pancreas-specific genes, which when mutated decreased linked chloramphenicol acetyltransferase expression up to 170-fold in 266-6 cells. This footprint may represent a binding site for one or more pancreas-specific regulatory proteins.     

Functional dissection of an enhancer-like element located within the second intron of the human <Emphasis Type="Italic">U2AF1L4</Emphasis> gene

A detailed functional and evolutionary analysis of an enhancer element of the human genome (enhancer 12) located in the second intron of the U2AF1L4 gene, which we identified earlier, is presented. Overlapping fragments of the studied genome region were analyzed for enhancer activity, and the site responsible for the activity of this element was identified using transient transfections of HeLa cells. Comparison of the enhancer 12 sequence with orthologous sequences from seven primate species revealed the existence of evolutionarily conserved sequences within this element. One of the identified conservative regions is likely responsible for the enhancer activity and is able to specifically interact in vitro with proteins of HeLa cell nuclear extract. The ability of orthologous primate sequences to compete with enhancer 12 for binding with HeLa cell nuclear extract proteins and to enhance the activity of the reporter gene in transient transfection of HeLa cells is demonstrated.     

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.     

Structure and function of the enhancer 3'' to the human A gamma globin gene.

An enhancer is located immediately 3' to the A gamma globin gene. We have used DNase I footprinting to map the sites of interaction of nuclear proteins with the DNA sequences of this enhancer. Eight footprints were discovered, distributed over 600 base pairs of DNA. Three of these contain a consensus binding site for the erythroid specific factor GATA-I. Each of these GATA-1 sites had an enhancer activity when inserted into a reporter plasmid and tested in human erythroleukemia cells. Other footprints within the enhancer contained consensus binding sequences for the ubiquitous, positive regulatory proteins AP2 and CBP-1. An Sp1-like recognition sequence was also identified. Synthetic oligonucleotides encompassing two of the footprints generated a slowly migrating complex in gel mobility shift assays. The same complex forms on a fragment of the human gamma globin gene promoter extending from -260 to -200. The DNaseI footprint of this protein complex with the enhancer overlapped a sequence, AGGAGGA, found within the binding site for a protein that interacts with the chicken beta globin promoter and enhancer, termed the stage selector element. We propose that this complex of proteins may be involved in the human gamma globin promoter-enhancer interaction.     

The mast cell-specific expression of a protease gene, RMCP II, is regulated by an enhancer element that binds specifically to mast cell trans-acting factors

The rat mast cell protease gene, RMCP II, is specifically expressed in the mucosal subclass of rat mast cells. We show here that the 5'-flanking region of this gene contains a mast cell-specific enhancer that directs preferential expression of a linked reporter gene (human growth hormone) transfected into rat basophilic leukemia cells. A DNA fragment containing the enhancer sequence is capable of binding specifically to mast cell nuclear trans-acting factors. The sequence of this enhancer element contains a region of homology to a consensus core sequence present in the enhancer region of the pancreatic protease genes.     

Purification and characterization of a human pancreas-specific antigen

A pancreas-specific antigen was identified by immunologic techniques and purified from saline extract of human pancreas. The purified pancreas-specific antigen was shown to be homogeneous by polyacrylamide gel electrophoresis under both denaturing and non-denaturing conditions. It had a molecular weight of 44000 as estimated by gel filtration or sodium dodecyl sulfate-gel electrophoresis, and a sedimentation coefficient of 3.4 S as analyzed by sucrose gradient centrifugation. Pancreas-specific antigen possessed an isoelectric point of 4.9 and migrated to alpha-beta region upon immunoelectrophoresis. By colorimetric assay procedures, pancreas-specific antigen exhibited no enzyme activity, such as amylase, protease, esterase, lipase, acid phosphatase, alkaline phosphatase peroxidase, deoxyribonuclease or ribonuclease. Immunoreactivity of pancreas-specific antigen was sensitive to proteolytic enzymes, perchloric acid and high temperature (70 degrees C, 10 min); but insensitive to neuraminidase or beta-glucosidase. Immunohistochemical staining revealed that pancreas-specific antigen was located in acinar cells of human pancreas. In addition, a higher concentration of pancreas-specific antigen was detected in pancreatic juice than in the saline extract of pancreas. This newly identified pancreas-specific antigen, therefore, may be a useful marker protein in physiological studies of pancreas and pancreatic secretion.     

Nuclear factor EF-1A binds to the adenovirus E1A core enhancer element and to other transcriptional control regions.

We have identified a cellular enhancer-binding protein, present in nuclear extracts prepared from human and rodent cells, that binds to the adenovirus E1A enhancer element I sequence. The factor has been termed EF-1A, for enhancer-binding factor to the E1A core motif. EF-1A was found to bind to two adjacent, related sequence motifs in the E1A enhancer region (termed sites A and B). The binding of EF-1A to these adjacent sites, or to synthetic dimerized sites of either motif, was cooperative. The cooperative binding of EF-1A to these sites was not subject to strict spacing constraints. EF-1A also bound to related sequences upstream of the E1A enhancer region and in the polyomavirus and adenovirus E4 enhancer regions. The EF-1A-binding region in the E1A enhancer stimulated expression of a linked gene in human 293 cells when multimerized. Based on the contact sites for EF-1A binding determined by chemical interference assays, this protein appears to be distinct from any previously characterized nuclear binding protein.