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

1987 ◽  
Vol 7 (1) ◽  
pp. 326-334
Author(s):  
L Osborn ◽  
M P Rosenberg ◽  
S A Keller ◽  
M H Meisler
Keyword(s):  
Transgenic Mice ◽  
Promoter Sequence ◽  
Single Copy ◽  
Pancreatic Amylase ◽  
Flanking Sequence ◽  
Amylase Gene ◽  
Base Pairs ◽  
Tissue Specific ◽  
Amylase Genes ◽  
Insulin Dependent

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.

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

1987 ◽  
Vol 7 (1) ◽  
pp. 326-334 ◽  
Author(s):  
L Osborn ◽  
M P Rosenberg ◽  
S A Keller ◽  
M H Meisler
Keyword(s):  
Transgenic Mice ◽  
Promoter Sequence ◽  
Single Copy ◽  
Pancreatic Amylase ◽  
Flanking Sequence ◽  
Amylase Gene ◽  
Base Pairs ◽  
Tissue Specific ◽  
Amylase Genes ◽  
Insulin Dependent

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.

scholarly journals Concerted evolution of human amylase genes.

1988 ◽  
Vol 8 (3) ◽  
pp. 1197-1205 ◽  
Author(s):  
D L Gumucio ◽  
K Wiebauer ◽  
R M Caldwell ◽  
L C Samuelson ◽  
M H Meisler
Keyword(s):  
Pancreatic Amylase ◽  
Amylase Gene ◽  
Salivary Amylase ◽  
Specific Expression ◽  
Enhancer Element ◽  
Tissue Specific ◽  
Tissue Specific Expression ◽  
Gene Copies ◽  
Amylase Genes ◽  
Sequence Elements

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.

Concerted evolution of human amylase genes

1988 ◽  
Vol 8 (3) ◽  
pp. 1197-1205
Author(s):  
D L Gumucio ◽  
K Wiebauer ◽  
R M Caldwell ◽  
L C Samuelson ◽  
M H Meisler
Keyword(s):  
Pancreatic Amylase ◽  
Amylase Gene ◽  
Salivary Amylase ◽  
Specific Expression ◽  
Enhancer Element ◽  
Tissue Specific ◽  
Tissue Specific Expression ◽  
Gene Copies ◽  
Amylase Genes ◽  
Sequence Elements

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.

Transcriptional insulation of the human keratin 18 gene in transgenic mice

1993 ◽  
Vol 13 (4) ◽  
pp. 2214-2223
Author(s):  
N Neznanov ◽  
I S Thorey ◽  
G Ceceña ◽  
R G Oshima
Keyword(s):  
Transgenic Mice ◽  
Thymidine Kinase ◽  
Copy Number ◽  
Thymidine Kinase Gene ◽  
Flanking Sequence ◽  
Kinase Gene ◽  
Tissue Specific ◽  
Dependent Behavior ◽  
Flanking Sequences ◽  
Keratin 18

Expression of the 10-kb human keratin 18 (K18) gene in transgenic mice results in efficient and appropriate tissue-specific expression in a variety of internal epithelial organs, including liver, lung, intestine, kidney, and the ependymal epithelium of brain, but not in spleen, heart, or skeletal muscle. Expression at the RNA level is directly proportional to the number of integrated K18 transgenes. These results indicate that the K18 gene is able to insulate itself both from the commonly observed cis-acting effects of the sites of integration and from the potential complications of duplicated copies of the gene arranged in head-to-tail fashion. To begin to identify the K18 gene sequences responsible for this property of transcriptional insulation, additional transgenic mouse lines containing deletions of either the 5' or 3' distal end of the K18 gene have been characterized. Deletion of 1.5 kb of the distal 5' flanking sequence has no effect upon either the tissue specificity or the copy number-dependent behavior of the transgene. In contrast, deletion of the 3.5-kb 3' flanking sequence of the gene results in the loss of the copy number-dependent behavior of the gene in liver and intestine. However, expression in kidney, lung, and brain remains efficient and copy number dependent in these transgenic mice. Furthermore, herpes simplex virus thymidine kinase gene expression is copy number dependent in transgenic mice when the gene is located between the distal 5'- and 3'-flanking sequences of the K18 gene. Each adult transgenic male expressed the thymidine kinase gene in testes and brain and proportionally to the number of integrated transgenes. We conclude that the characteristic of copy number-dependent expression of the K18 gene is tissue specific because the sequence requirements for transcriptional insulation in adult liver and intestine are different from those for lung and kidney. In addition, the behavior of the transgenic thymidine kinase gene in testes and brain suggests that the property of transcriptional insulation of the K18 gene may be conferred by the distal flanking sequences of the K18 gene and, additionally, may function for other genes.

scholarly journals Distinct regions control transcriptional activation of the alpha1(VI) collagen promoter in different tissues of transgenic mice.

1996 ◽  
Vol 135 (4) ◽  
pp. 1163-1177 ◽  
Author(s):  
P Braghetta ◽  
C Fabbro ◽  
S Piccolo ◽  
D Marvulli ◽  
P Bonaldo ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Transcriptional Activation ◽  
Promoter Sequence ◽  
Regulation Of Transcription ◽  
Flanking Sequence ◽  
Endogenous Gene ◽  
Intervertebral Disks ◽  
Specific Regulation ◽  
Beta Galactosidase ◽  
Different Tissues

To identify regions involved in tissue specific regulation of transcription of the alpha1(VI) collagen chain, transgenic mice were generated carrying various portions of the gene's 5'-flanking sequence fused to the E. coli beta-galactosidase gene. Analysis of the transgene expression pattern by X-gal staining of embryos revealed that: (a) The proximal 0.6 kb of promoter sequence activated transcription in mesenchymal cells at sites of insertion of superficial muscular aponeurosis into the skin; tendons were also faintly positive. (b) The region between -4.0 and -5.4 kb from the transcription start site was required for activation of the transgene in nerves. It also drove expression in joints, in intervertebral disks, and in subepidermal and vibrissae mesenchyme. (c) The fragment comprised within -6.2 and -7.5 kb was necessary for high level transcription in skeletal muscle and meninges. Positive cells in muscle were mostly mononuclear and probably included connective tissue elements, although staining of myoblasts was not ruled out. This fragment also activated expression in joints, in intervertebral disks, and in subepidermal and vibrissae mesenchyme. (d) beta-Galactosidase staining in vibrissae induced by the sequences -4.0 to -5.4 and -6.2 to -7.5 was not coincident: with the latter sequence labeled nuclei were found mainly in the ventral and posterior quadrant, and, histologically, in the outer layers of mesenchyme surrounding and between the follicles, whereas with the former the remaining quadrants were positive and expressing cells were mostly in the inner layers of the dermal sheath. (e) Other tissues, notably lung, adrenal gland, digestive tract, which produce high amounts of collagen type VI, did not stain for beta-galactosidase. (f) Central nervous system and retina, in which the endogenous gene is inactive, expressed the lacZ transgene in most lines. The data suggest that transcription of alpha1(VI) in different tissues is regulated by distinct sequence elements in a modular arrangement, a mechanism which confers high flexibility in the temporal and spatial pattern of expression during development.

Dietary regulation of pancreatic amylase in transgenic mice mediated by a 126-base pair DNA fragment

1992 ◽  
Vol 262 (6) ◽  
pp. G971-G976 ◽  
Author(s):  
R. M. Schmid ◽  
M. H. Meisler
Keyword(s):  
Transgenic Mice ◽  
Base Pair ◽  
Insulin Response ◽  
Regulatory Elements ◽  
Regulatory Sequences ◽  
Pancreatic Amylase ◽  
Amylase Gene ◽  
Dietary Regulation ◽  
Dna Fragment ◽  
Responsive Element

Expression of the mouse pancreatic amylase gene Amy-2.2 is increased approximately 10-fold in response to increasing the carbohydrate content of the diet from 9.6 to 74%. The DNA sequence mediating this response has been localized to the 5' flanking region of the amylase gene by analysis of hybrid constructs in transgenic mice. The results define a 127-base pair dietary response unit that includes two previously described regulatory elements, an insulin-responsive element and a pancreatic enhancer. Fragments containing these two elements alone fail to respond to diet, demonstrating a requirement for additional regulatory sequences. Another mouse amylase gene Amy-2.1 is only minimally responsive to insulin and to diet. The data are consistent with the hypothesis that the insulin-response element is necessary but not sufficient for regulation of amylase by dietary carbohydrate.

scholarly journals The effects of mutations in the ant promoter of phage P22 depend on context.

Genetics ◽  
1988 ◽  
Vol 120 (2) ◽  
pp. 319-327
Author(s):  
D Graña ◽  
T Gardella ◽  
M M Susskind
Keyword(s):  
Context Effects ◽  
Promoter Sequence ◽  
Single Copy ◽  
Promoter Strength ◽  
Lacz Gene ◽  
Promoter Mutation ◽  
Base Pairs ◽  
Phage P22 ◽  
Beta Galactosidase

Abstract Recombination was used to construct 22 two- or three-way combinations of down- and up-mutations in Pant, a strong, near-consensus promoter of phage P22. The relative strengths of these promoters in vivo were assayed by fusing them to an ant/lacZ gene fusion and measuring beta-galactosidase levels produced by lysogens carrying the fusions on single-copy prophages. The results of these assays show that the magnitude of the effect of a promoter mutation can vary considerably when its context is changed by the presence of another mutation. In addition, as Pant approaches conformity with the consensus promoter sequence, the up-mutations decrease promoter strength, even though the same mutations increase promoter strength in the presence of a down-mutation. These context effects imply that individual consensus base pairs cannot be considered to contribute to promoter strength independently.

scholarly journals An upstream regulatory region mediates high-level, tissue-specific expression of the human alpha 1(I) collagen gene in transgenic mice.

1991 ◽  
Vol 11 (4) ◽  
pp. 2066-2074 ◽  
Author(s):  
J L Slack ◽  
D J Liska ◽  
P Bornstein
Keyword(s):  
Transgenic Mice ◽  
Regulatory Region ◽  
Collagen Gene ◽  
Flanking Sequence ◽  
Specific Expression ◽  
Tissue Specific ◽  
Endogenous Gene ◽  
Tissue Specific Expression ◽  
First Intron ◽  
High Level

Studies in vitro have not adequately resolved the role of intronic and upstream elements in regulating expression of the alpha 1(I) collagen gene. To address this issue, we generated 12 separate lines of transgenic mice with alpha 1(I) collagen-human growth hormone (hGH) constructs containing different amounts of 5'-flanking sequence, with or without most of the first intron. Transgenes driven by 2.3 kb of alpha 1(I) 5'-flanking sequence, whether or not they contained the first intron, were expressed at a high level and in a tissue-specific manner in seven out of seven independent lines of transgenic mice. In most tissues, the transgene was expressed at levels approaching that of the endogenous alpha 1(I) gene and was regulated identically with the endogenous gene as animals aged. However, in lung, expression of the transgene was anomalously high, and in muscle, expression was lower than that of the endogenous gene, suggesting that in these tissues other regions of the gene may participate in directing appropriate expression. Five lines of mice were generated containing transgenes driven by 0.44 kb of alpha 1(I) 5'-flanking sequence (with or without the first intron), and expression was detected in four out of five of these lines. The level of expression of the 0.44-kb constructs in the major collagen-producing tissues was 15- to 500-fold lower than that observed with the longer 2.3-kb promoter. While transgenes containing the 0.44-kb promoter and the first intron retained a modest degree of tissue-specific expression, those without the first intron lacked tissue specificity and were poorly expressed in all tissues except lung.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Genomic Regions that Mediate Placental Cell-Specific and Developmental Regulation of Human Cyp19 (Aromatase) Gene Expression in Transgenic Mice

Endocrinology ◽  
2005 ◽  
Vol 146 (5) ◽  
pp. 2481-2488 ◽  
Author(s):  
Amrita Kamat ◽  
Margaret E. Smith ◽  
John M. Shelton ◽  
James A. Richardson ◽  
Carole R. Mendelson
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Developmental Regulation ◽  
Giant Cells ◽  
Regulatory Elements ◽  
Deletion Mapping ◽  
Flanking Sequence ◽  
Specific Expression ◽  
Tissue Specific ◽  
Cyp19 Gene

Abstract The human aromatase (hCYP19) gene is controlled by tissue-specific promoters that lie upstream of tissue-specific first exons. Placenta-specific exon I.1 lies approximately 100,000 bp upstream of exon II. Previously, we observed that genomic sequences within 501 bp upstream of exon I.1 mediate placenta-specific expression. In the present study, transgenic mice were created carrying hCYP19I.1−246:hGH/hGX, hCYP19I.1−201:hGH, and hCYP19I.1−125:hGH fusion genes to further delineate 5′-flanking sequences within 501 bp of exon I.1 that are required to mediate placenta-specific hCYP19 gene expression. As little as 246 bp of hCYP19 exon I.1 5′-flanking sequence was sufficient to direct placenta-specific expression in transgenic mice. By contrast, transgenes containing 201 or 125 bp of exon I.1 5′-flanking DNA were not expressed in mouse placenta. Furthermore, hCYP19I.1−246:hGX transgene expression was developmentally regulated; expression was observed as early as embryonic d 7.5 (E7.5) in several cells of the trophoblast ectoderm, on E8.5 in some trophoblast giant cells, and by E9.5 in giant cells and the labyrinthine layer. By contrast, expression of the hCYP19I.1−501:hGH transgene was first observed on E10.5 and was restricted to the labyrinthine layer, which is most analogous to the human syncytiotrophoblast. This suggests the presence of regulatory elements between −501 and −246 bp that may bind inhibitory transcription factors expressed in giant cells. These findings from transgenic experiments together with deletion mapping studies using transfected human placental cells indicate that the concerted interaction of strong placenta-specific enhancers and silencers within this 501-bp region mediate labyrinthine and syncytiotrophoblast-specific CYP19 gene expression.

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