Growth Hormone Fusion Gene Research Articles

Glucocorticoid inhibition of SP-A gene expression in lung type II cells is mediated via the TTF-1-binding element.

Induction of surfactant protein-A (SP-A) gene expression in fetal lung type II cells by cAMP and IL-1 is mediated by increased binding of thyroid transcription factor-1 (TTF-1) and NF-B proteins p50 and p65 to the TTF-1-binding element (TBE) at -183 bp. In type II cell transfections, dexamethasone (Dex) markedly inhibits cAMP-induced expression of rabbit SP-A:human growth hormone (hGH) fusion genes containing as little as 300 bp of the SP-A 5'-flanking sequence. Dex inhibition is blocked by RU-486, suggesting a role of the glucocorticoid receptor (GR). The present study was undertaken to define the mechanisms for GR inhibition of SP-A expression. Cotransfection of primary cultures of type II cells with a GR expression vector abrogated cAMP induction of SP-A promoter activity while, at the same time, causing a 60-fold induction of cotransfected mouse mammary tumor virus (MMTV) promoter. In lung cells transfected with a fusion gene containing three TBEs fused to the basal SP-A promoter, Dex prevented the stimulatory effect of IL-1 on TTF-1 induction of SP-A promoter activity, suggesting that the GR inhibits SP-A promoter activity through the TBE. In gel shift assays using nuclear extracts from human fetal type II cells cultured in the absence or presence of cAMP, Dex markedly reduced binding of nuclear proteins to the TBE and blocked the stimulatory effect of cAMP on TBE-binding activity. Our finding that Dex increased expression of the NF-kappaB inhibitory partner IkappaB-alpha suggests that the decrease in TBE-binding activity may be caused, in part, by GR inhibition of NF-kappaB interaction with this site.

Cyclic AMP-responsive Expression of the Surfactant Protein-A Gene Is Mediated by Increased DNA Binding and Transcriptional Activity of Thyroid Transcription Factor-1

Surfactant protein (SP)-A gene transcription is stimulated by factors that increase cyclic AMP. In the present study, we observed that three thyroid transcription factor-1 (TTF-1) binding elements (TBEs) located within a 255 base pair region flanking the 5'-end of the baboon SP-A2 (bSP-A2) gene are required for maximal cyclic AMP induction of bSP-A2 promoter activity. We found that TTF-1 DNA binding activity was increased in nuclear extracts of pulmonary type II cells cultured in the presence of cyclic AMP. By contrast, the levels of immunoreactive TTF-1 protein were similar in nuclear extracts of control and cyclic AMP-treated type II cells. The incorporation of [32P]orthophosphate into immunoprecipitated TTF-1 protein also was markedly increased by cyclic AMP treatment. Moreover, exposure of nuclear extracts from cyclic AMP-treated type II cells either to potato acid phosphatase or alkaline phosphatase abolished the cyclic AMP-induced increase in TTF-1 DNA-binding activity. Interestingly, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), known to activate protein kinase C, also enhanced incorporation of [32P]orthophosphate into TTF-1 protein; however, the DNA binding activity of TTF-1 was decreased in nuclear extracts of TPA-treated type II cells. Expression vectors encoding TTF-1 and the catalytic subunit of protein kinase A (PKA-cat) were cotransfected into A549 lung adenocarcinoma cells together with an SPA:human growth hormone fusion gene (255 base pairs of 5'-flanking DNA from the baboon SP-A2 gene linked to human growth hormone, as reporter) containing TBEs, or with a reporter gene construct containing three tandem TBEs fused upstream of the bSP-A2 gene TATA box and the transcription initiation site. Coexpression of TTF-1 and PKA-cat increased fusion gene expression 3-4-fold as compared with expression of TTF-1 in the absence of PKA-cat. Moreover, the transcriptional activity of TTF-1 was suppressed by cotransfection of a dominant negative form of PKA regulatory subunit RIalpha. We suggest that a PKA-induced increase of TTF-1 phosphorylation and TBE binding activity mediates cyclic AMP-induced expression of the SP-A gene in lung type II cells.

Suppressor and Activator Functions Mediated by a Repeated Heptad Sequence in the Liver Fatty Acid-binding Protein Gene (Fabpl): EFFECTS ON RENAL, SMALL INTESTINAL, AND COLONIC EPITHELIAL CELL GENE EXPRESSION IN TRANSGENIC MICE

A 35-nucleotide sequence in the liver fatty acid-binding protein gene (Fabpl) has been identified that interacts with nuclear proteins present in adult mouse liver, kidney, stomach, small intestine, and colon. The binding site consists of a direct heptad repeat (TTCTGNNTT) separated by five nucleotides. Both heptads are required for formation of stable complexes with nuclear proteins in gel mobility shift assays. The in vivo functions mediated by the repeats were determined by comparing the expression of four Fabpl/human growth hormone fusion genes in multiple pedigrees of adult transgenic mice. The transgenes contained (i) nucleotides -596 to +21 of Fabpl linked to the human growth hormone reporter, (ii) 4 additional copies of the 35-base pair element placed at nucleotide -596 of Fabpl, (iii) 4 additional copies of the sequence placed just upstream of its endogenous site at nucleotide -132, and (iv) a sequence identical to (iii) but with all heptad repeats mutated within each of the 4 additional copies of the 35-base pair element. Transgene expression was defined by RNA blot hybridizations and by light and electron microscopic immunohistochemistry. The heptad repeat functions to suppress expression in tubular epithelial cells of the proximal nephron, in hepatocytes, in the mucus-producing pit cells of the gastric epithelium, and in absorptive enterocytes located in the proximal small intestine. There is a gradient of escape from enterocytic suppression as one moves from the proximal to distal small intestine. This escape progresses to involve successively less differentiated cells located closer and closer to the stem cell zone in crypts of Lieberkühn. The heptad repeat activates gene expression in the colonic epithelium so that all proliferating and nonproliferating cells in colonic crypts distributed from the cecum to the rectum support transgene expression. The heptad has no obvious sequence similarities to known transcription factor binding sites, suggesting that mediators of its in vivo activities are likely to be novel. One candidate factor is a 90-kDa protein identified in Southwestern blots. The 90-kDa protein also binds to an element in the matrix metalloproteinase-2 gene that functions as an enhancer in renal cells, shares sequence homology with the heptad, and generates similar-sized complexes in gel mobility shift assays as the Fabpl repeat. The heptad repeat represents a target for identifying transcription factors that regulate gene expression between gut and renal epithelia and that also regulate the differentiation program of the intestine's principal epithelial lineage as a function of its location along the duodenal-colonic axis. Finally, the Fabpl regulatory elements described in this report should be useful for delivering a variety of gene products throughout the colonic epithelium of transgenic mice.

Transfer of an ovine metallothionein-ovine growth hormone fusion gene into swine.

An ovine metallothionein-1a (oMT1a)-ovine growth hormone (oGH) fusion gene was microinjected into 400 pig zygotes, the zygotes were transferred into recipient females, and 15 founder transgenic pigs were born. Of 12 transgenic pigs assayed, five expressed high levels of oGH (> 900 ng/mL plasma), one expressed low levels of oGH (10 to 30 ng/mL), and six did not express oGH. Dietary supplementation with 2,000 ppm of zinc for 6 d induced a 20-fold increase in plasma oGH in the transgenic pig with low expression but did not induce expression in the six transgenic pigs with no constitutive oGH expression. The average daily gain of five transgenic pigs with elevated oGH was similar to that of non-transgenic littermates during a 9-wk feeding trial (P = .52). The liver, kidney, adrenal, and thyroid weights were all significantly heavier for the oGH-expressing transgenic pigs than for non-transgenic littermates. Total carcass fat, longissimus muscle fat, subcutaneous backfat thickness, and loin eye area were lower and carcass protein and water content and beta R fiber area of longissimus muscle were higher in the transgenic pigs with elevated oGH than in their littermate controls (P < .05 for each). The data indicate that even though the oMT1a promoter was more inducible by zinc than was previously reported for the mouse MT promoter in swine, the former provided a higher level of oGH expression than the mouse MT promoter.

Microencapsulation and transplantation of genetically engineered cells: a new approach to somatic gene therapy.

In order to develop a model for gene therapy which avoids dependence on an autologous source of target cells and immunosuppressive therapy, mouse Ltk fibroblasts transfected with a human growth hormone (hGH) fusion gene were encapsulated in a semipermeable alginate-poly-L-lysine-alginate (APA) membrane. The encapsulated cells were cultured in vitro or transplanted intraperitoneally into mice to monitor cell viability, cell growth, and hGH secretion. The effect of Zn2+ ions on vector expression was also monitored in vitro and in vivo. Results indicate that: (1) the capsule environment is compatible with cell viability and cell growth; (2) the capsule limits cell growth; (3) the capsule membrane is permeable to the exit of hGH; (4) gene product expression may be stimulated by external means; (5) the novel gene product is delivered in vivo; and (6) encapsulated cells recovered from transplant recipients continue to secrete hGH in vitro. The results suggest therapeutic potential of this approach to somatic gene therapy.

Autofeedback suppression of growth hormone (GH) secretion in transgenic mice expressing a human GH reporter targeted by tyrosine hydroxylase 5'-flanking sequences to the hypothalamus.

Transgenic mice expressing a tyrosine hydroxylase-human (h) GH fusion gene in the hypothalamus exhibit a dwarf phenotype. The GH feedback mechanism(s) underlying the growth retardation in these animals was investigated by assessing peptide and messenger RNA (mRNA) levels of the hormones of the hypothalamic-GH-IGF-I axis. Pituitary GH content, hypothalamic GH-releasing hormone (GHRH) and somatostatin (SRIH) content, and serum IGF-I levels were measured by RIA. mRNA levels of hypothalamic GHRH and SRIH and of pituitary GH and the GHRH receptor were measured by Northern blot hybridization. Transgenic mice of both sexes and their wild-type littermates were studied at 2-4 months of age. The pituitary GH content was markedly reduced by 85% in male and by 87% in female transgenic mice compared to that in wild-type controls (P < 0.01 for both). The pituitary GH mRNA content was also decreased by 73% (P = 0.002) in transgenic male mice. Circulating IGF-I levels were significantly reduced by 66% and 68% in male and female transgenic mice, respectively (P = 0.001). The hypothalamic GHRH content was significantly reduced by 19% and 33% (P < 0.05) in male and female transgenic mice, respectively. No significant difference was detected, however, in the hypothalamic SRIH content between wild-type and transgenic mice. Hypothalamic GHRH mRNA levels were significantly decreased by 35% (P = 0.002) in transgenic male mice compared to those in wild-type littermates. In contrast, SRIH mRNA was not significantly changed. An even greater reduction (61%; P = 0.003) was observed in pituitary GHRH receptor mRNA in transgenic mice. These data indicate that the GH deficiency and dwarf phenotype of the tyrosine hydroxylase-hGH transgenic mouse can be attributed primarily to impaired hypothalamic GHRH production. The mechanism of GH feedback inhibition appears to involve direct suppression of GHRH gene expression by locally produced hGH in the hypothalamus.

Methylation and expression of a metallothionein promoter ovine growth hormone fusion gene (MToGH1) in transgenic mice

We have examined transgene methylation in the DNA from the livers of a pedigree of mice carrying three copies of an integrated MToGH1 transgene. Utilizing the methylation-sensitive isoschizomers Msp I and Hpa II, Southern blot analysis revealed that all second generation animals derived from a transgenic female had hypermethylated DNA, whereas first generation animals sired by a transgenic male displayed a range of methylation phenotypes ranging from no methylation to hypermethylation of the transgene sequences. Of the mice that exhibited hypermethylation of the transgene in CpG dinucleotides (CmCGG), a minority of these animals also exhibited apparent CpC methylation (i.e. inhibition of Msp I cutting, presumably blocked by methylation of the outer C of CCGG). Methylation was also examined in the inner C of CC(A/T)GG sequences in the MToGH1 transgene using the isoschizomer pair BstN I and EcoR II. A minority of MToGH1 animals in the F1 generation showed clear evidence of methylation in these sites as well as in the inner and outer Cs of CCGG sites. An examination of MToGH1 expression in terms of oGH levels in serum revealed that there was a high degree of variation in the levels of circulating oGH between animals of this pedigree. There was a weak inverse relationship between the serum level of oGH and the extent of methylation of the transgene. In particular, mice exhibiting CpC together with CpG methylation were found to have very low levels of circulating oGH. Our results highlight the nature and complexity of epigenetic factors associated with transgene sequences which may ultimately influence expression of introduced genes in the mammalian genome.

Directed endothelial differentiation of cultured embryonic yolk sac cells in vivo provides a novel cell-based system for gene therapy.

Cultured murine yolk sac cells transfected with the cytomegalovirus immediate early promoter/human growth hormone (CMVIE-hGH) fusion gene, expressing high levels of hGH in culture, and suspended in Matrigel were subcutaneously (s.c.) injected into experimental mice. The injected cells were shown to form discrete vesicular structures within the Matrigel implant, suggesting directed differentiation of the embryonic yolk sac cells into endothelial tissue. Human growth hormone radioimmune assay of these mice showed sustained physiologically significant levels of hGH in their serum for beyond four months. These results confirmed that long-term cultured murine embryonic yolk sac cells can be induced to differentiate into endothelial cells both in vivo and in vitro and suggested a novel approach to the delivery to the circulation of therapeutic proteins for the treatment of inherited and acquired diseases.

Rescue of pregnancy and maintenance of corpora lutea in infertile transgenic mice expressing an ovine metallothionein 1a-ovine growth hormone fusion gene.

Transgenic female mice expressing a temporally regulatable ovine metallothionein 1a-ovine growth hormone (oMT1a-oGH) fusion gene can cycle, mate, and support early embryonic development; but they fail to maintain pregnancy or, in most cases, to exhibit signs of pseudopregnancy. The present study was designed to determine whether or not the infertility of oMT1a-oGH transgenic female mice is due to luteal insufficiency. A series of five experiments was conducted in which various hormonal therapies were utilized in an attempt to overcome this infertility. Provision of 1 mg progesterone to oMT1a-oGH females from Days 1 to 17 of gestation rescued pregnancy in 19 of 20 females with an increased mean litter size (p < 0.01) compared to that of wild-type control females. Supplementation with progesterone also appeared on the basis of gross anatomical appearance to maintain CI. Provision of 2 mg progesterone in combination with 25 ng estradiol-17 beta from Days 4 to 17 rescued pregnancy in 14 of 24 oMT1a-oGH females, while provision of 100 or 25 micrograms ovine prolactin from Days 3 to 17 failed to overcome oMT1a-oGH infertility. Transgenic females with rescued pregnancies lactated and raised heavier pups than wild-type females by Day 15 postnatal (p < 0.05). Results indicate that infertility of oMT1a-oGH transgenic mice is caused by luteal insufficiency. Supplementation of transgenic females with progesterone appeared to maintain morphologically normal CL and pregnancy with subsequently increased litter size and normal lactation.

Effects of glucose on insulin secretion, glucokinase activity, and transgene expression in transgenic mouse islets containing an upstream glucokinase promoter-human growth hormone fusion gene

Effects of glucose on insulin secretion, glucokinase activity, and transgene expression in transgenic mouse islets containing an upstream glucokinase promoter-human growth hormone fusion gene

Effects of Glucose on Insulin Secretion, Glucokinase Activity, and Transgene Expression in Transgenic Mouse Islets Containing an Upstream Glucokinase Promoter-Human Growth Hormone Fusion Gene

We have analyzed in organ culture the effects of glucose on glucose-induced insulin secretion, glucokinase (GK) activity, and human growth hormone (hGH) expression in pancreatic islets from transgenic mice containing an upstream GK promoter-hGH fusion gene. Freshly isolated islets from these mice had a normal insulin secretory response to glucose but showed subtle defects after culture in low or high glucose for 4 days that may have been due to the accumulation of hGH in the culture media. Islets cultured from both normal and transgenic mice had approximately a fourfold induction of GK activity in response to an increased concentration of glucose in the culture media, whereas no such change in total islet hGH production was observed. Immunocytochemical localization of hGH in islets cultured in 3 mM glucose showed a pattern similar to that in freshly isolated islets. However, after culture in 30 mM glucose, hGH immunostaining became strikingly more heterogeneous. We conclude 1) that GK-hGH transgene expression does not appear to adversely affect glucose-stimulated insulin secretion in vivo or in freshly isolated islets, 2) that glucose does not induce transgene expression, thus providing additional evidence against an effect of glucose on GK gene transcription in the islet, and 3) that glucose stimulates the co-release of hGH with insulin, thereby enhancing the heterogeneous staining pattern seen among pancreatic beta-cells.

Expression of an ovine growth hormone transgene in mice increases archidonic acid in cellular membranes

The degree of unsaturation of membrane lipids has been implicated in a number of physiological disorders, yet its regulation remains poorly understood, especially the regulation of the synthesis and distribution of arachidonic acid levels, the most abundant long chain polyunsaturated fatty acid in membranes. Transgenic mice expressing the ovine metallothionein 1a-ovine growth hormone (oMt1a-oGH) fusion gene exhibited significantly elevated levels of a number of long chain polyunsaturated fatty acids in serum, including arachidonic acid. In oMt1a-oGH transgenic mice the products of all three desaturation pathways are affected by the expression of the ovine growth hormone transgene. The essential precursors of membrane long chain polyunsaturated fatty acids, 18:2n-6 and 18:3n-3, were reduced in transgenics relative to controls, and their desaturation and elongation products, arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3), were elevated. As rare intermediate long chain polyunsaturated fatty acids such as eicosatrienoic acid (20:3n-9) were also significantly elevated, we conclude that these observations reflect increased activity of the delta-5 and delta-6 desaturase enzymes. In contrast, the products of the stearoyl CoA or delta-9 desaturase, were significantly reduced in oMt1a-oGH expressing transgenics relative to their levels in control mice.

Comparison of the patterns of expression of rat intestinal fatty acid binding protein/human growth hormone fusion genes in cultured intestinal epithelial cell lines and in the gut epithelium of transgenic mice

The intestinal fatty acid binding protein gene (Fabpi) provides a good model system for studying how gene transcription is regulated in enterocytes as a function of their differentiation program and location along the duodenal-to-colonic axis. We have compared and contrasted the transcriptional activity of four fusion genes composed of elements from the 5'-nontranscribed domain of rat Fabpi linked to the human growth hormone gene (I-FABP/hGH) in transgenic mice and in five primate epithelial cell lines derived from intestine, liver, kidney, and cervix. Nucleotides -103 to +28 of rat Fabpi are able to direct appropriate lineage-specific and geographic patterns of hGH expression in transgenic mice. I-FABP-103 to +28/hGH is preferentially expressed in Caco-2 cells, which emulate some of the features of differentiated small intestinal enterocytes after they reach confluence. However, other I-FABP/hGH fusion genes that exhibit differentiation-dependent changes in their expression along the crypt-to-villus axis do not manifest the same pattern of differentiation-dependent change in activity in this cell line. Correlation of their patterns of expression in vivo and ex vivo suggest that nonproliferating Caco-2 cells mimic some of the features of the transcriptional regulatory environment of enterocytes located in the upper crypt. Nucleotides -103 to +28 of rat Fabpi contain one copy of a repeated 14-base pair element that is conserved in the orthologous mouse and human genes and represented in several other homologous and nonhomologous genes, which are expressed in villus-associated enterocytes. This element binds to two members of the steroid hormone receptor superfamily of transcription factors produced in enterocytes and Caco-2 cells: hepatic nuclear factor-4 (HNF-4) and apolipoprotein regulatory protein-1 (ARP-1). Co-transfection studies performed in Caco-2 cells and in a monkey kidney cell line (CV-1) that lacks endogenous pools of ARP-1 and HNF-4 suggest that ARP-1 and HNF-4 can function to activate I-FABP-103 to +28/hGH+3 through their interactions with the 14-base pair element. This activation appears to be affected by elements located between nucleotides -277 and -104 and other transcription factors.

Expression of Human Growth Hormone Fusion Genes in Cultured Lung Endothelial Cells and in the Lungs of Mice

We sought to develop genetic therapy for acute lung diseases by introducing genes into lung cells in vivo that were only transiently expressed. To that end, we introduced a gene encoding a physiologically relevant secreted human protein into bovine lung endothelial cells in culture and into the lungs of mice using the technique of lipofection. We exposed cultured endothelial cells to a plasmid containing the coding region for human growth hormone (hGH) driven by a metallothionein (MT) promoter. In cells lipofected with the plasmid containing the MT promoter, expression of the hGH gene in medium was low (peak = 30 ng hGH/24 h/60-mm dish), but expression was markedly increased by addition of either dexamethasone (peak = 91) or cadmium (peak = 120). Lipofection with the same construct except a thymidine kinase promoter showed no cadmium response. We gave mice 5,000 ppm ZnSO4 in their drinking water and 24 h later injected intravenously plasmid containing the MT promoter complexed to liposomes. Mice were killed 1, 3, and 5 days after injection, and hGH production by minced lung, liver, and kidneys was determined in vitro. Neither kidneys nor liver produced detectable hGH. However, hGH was produced by the lungs, beginning on day 1, peaking on day 3 (approximately 1.0 ng hGH/24 h/g tissue), and declining by day 5. Lungs from mice injected either with DNA alone or with liposome alone did not produce hGH. mRNA specific for hGH was demonstrated in the lungs by polymerase chain reaction amplification of cDNA followed by agarose gel electrophoreses.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous localization of six antigens in single sections of transgenic mouse intestine using a combination of light and fluorescence microscopy.

To study the geographic differentiation of the intestinal epithelium and to understand the complex lineage relationships of its cell populations, it is often necessary to visualize the protein products of multiple genes in sections prepared from different positions along the duodenal-to-colonic and/or crypt-to-villus axes. Multilabel fluorescence or brightfield immunohistochemical techniques have previously been used for this purpose. However, the number of antigens that can be identified on single sections is limited in fluorescence microscopy by the number of fluorophores with non-overlapping absorption and emission characteristics, in brightfield microscopy by the number of visually distinguishable chromogens, and in both methods by the availability of primary antisera raised in multiple species. We have now used a combination of light and fluorescence microscopic techniques to increase the number of antigens that can be detected in a single section to six. Sections were sequentially stained using immunogold with silver intensification, peroxidase-antiperoxidase with diaminobenzidine chromogen, and peroxidase-anti-peroxidase with alpha-naphthol/basic dye as chromogen, followed by simultaneous fluorescent detection with fluorescein, 7-amino-4-methylcoumarin-3-acetic acid, and beta-phycoerythrin. This method enables up to four separate antigens to be visualized within a single cell and two additional antigens to be detected in unrelated cells. The technique is illustrated by examining the cellular patterns of expression of liver fatty acid binding protein/human growth hormone fusion genes in the intestinal epithelium of adult transgenic mice. It should be generally applicable to other experimental systems that require localization of multiple antigens in single tissue sections.

Transgenic female mice with high human growth hormone levels are fertile and capable of normal lactation without having been pregnant.

Transgenic mice carrying the phosphoenolpyruvate carboxykinase promoter region-human growth hormone (PEPCK-hGH) fusion gene are characterized by accelerated growth and plasma hGH levels ranging from 100 to 700 ng/ml. Both sexes are fertile, in contrast to previous findings in metallothionein-I/hGH transgenic mice in which females are sterile, apparently due to luteal failure. Virgin transgenic PEPCK/hGH females from this line produce milk and can successfully raise foster litters to weaning. We conclude that the life-long presence of very large amounts of hGH in the circulation is compatible with ovulation, can override the effects of hGH-induced suppression of endogenous PRL release, and can support full lactation in animals that have not been primed by hormonal changes associated with pregnancy.

Tissue-specific, developmental, hormonal, and dietary regulation of rat phosphoenolpyruvate carboxykinase-human growth hormone fusion genes in transgenic mice.

The cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene is expressed in multiple tissues and is regulated in a complex tissue-specific manner. To map the cis-acting DNA elements that direct this tissue-specific expression, we made transgenic mice containing truncated PEPCK-human growth hormone (hGH) fusion genes. The transgenes contained PEPCK promoter fragments with 5' endpoints at -2088, -888, -600, -402, and -207 bp, while the 3' endpoint was at +69 bp. Immunohistochemical analysis showed that the -2088 transgene was expressed in the correct cell types (hepatocytes, proximal tubular epithelium of the kidney, villar epithelium of the small intestine, epithelium of the colon, smooth muscle of the vagina and lungs, ductal epithelium of the sublingual gland, and white and brown adipocytes). Solution hybridization of hGH mRNA expressed from the transgenes indicated that white and brown fat-specific elements are located distally (-2088 to -888 bp) and that liver-, gut-, and kidney-specific elements are located proximally (-600 to +69 bp). However, elements outside of the region tested are necessary for the correct developmental pattern and level of PEPCK expression in kidney. Both the -2088 and -402 transgenes responded in a tissue-specific manner to dietary stimuli, and the -2088 transgene responded to glucocorticoid stimuli. Thus, different tissues utilize distinct cell-specific cis-acting elements to direct and regulate the PEPCK gene.

Tissue-specific, developmental, hormonal, and dietary regulation of rat phosphoenolpyruvate carboxykinase-human growth hormone fusion genes in transgenic mice.

The cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene is expressed in multiple tissues and is regulated in a complex tissue-specific manner. To map the cis-acting DNA elements that direct this tissue-specific expression, we made transgenic mice containing truncated PEPCK-human growth hormone (hGH) fusion genes. The transgenes contained PEPCK promoter fragments with 5' endpoints at -2088, -888, -600, -402, and -207 bp, while the 3' endpoint was at +69 bp. Immunohistochemical analysis showed that the -2088 transgene was expressed in the correct cell types (hepatocytes, proximal tubular epithelium of the kidney, villar epithelium of the small intestine, epithelium of the colon, smooth muscle of the vagina and lungs, ductal epithelium of the sublingual gland, and white and brown adipocytes). Solution hybridization of hGH mRNA expressed from the transgenes indicated that white and brown fat-specific elements are located distally (-2088 to -888 bp) and that liver-, gut-, and kidney-specific elements are located proximally (-600 to +69 bp). However, elements outside of the region tested are necessary for the correct developmental pattern and level of PEPCK expression in kidney. Both the -2088 and -402 transgenes responded in a tissue-specific manner to dietary stimuli, and the -2088 transgene responded to glucocorticoid stimuli. Thus, different tissues utilize distinct cell-specific cis-acting elements to direct and regulate the PEPCK gene.

The Mouse Intestinal Fatty Acid Binding Protein Gene: Nucleotide Sequence, Pattern of Developmental and Regional Expression, and Proposed Structure of Its Protein Product

The rat intestinal fatty acid binding protein (I-FABP) gene has been used as a model to study temporal and spatial differentiation of the gut epithelium while its protein product has been used as a model for examining the atomic details of noncovalent fatty acid-protein interactions. We have isolated the mouse I-FABP gene (Fabpi) and determined its nucleotide sequence. Comparisons of the orthologous mouse, rat, and human I-FABP genes revealed three conserved domains in their otherwise divergent 5' nontranscribed sequences. RNA blot hybridization and multilabel immunocytochemical methods were used to compare the developmental stage-specific patterns of activation of the rat and mouse genes. In addition, Fabpi expression in enterocytes was examined as a function of their differentiation along the crypto-to-villus and duodenal-to-colonic axes of the small intestine. Based on the similar temporal and geographic patterns of mouse and rat I-FABP expression described here and the results of our earlier studies of transgenic mice containing rat Fabpi/human growth hormone fusion genes, we propose that one of the conserved domains, spanning nucleotides -500 to -419 in mouse Fabpi, and/or a 14-bp element, are necessary for establishing and maintaining its region-specific expression along the duodenal-to-colonic axis of the perpetually renewing gut epithelium. Finally, predictions of the structure of mouse I-FABP using the refined 2.0 A model of rat I-FABP, suggest that a proline found at position 69 of the mouse, but not rat, protein may affect its ligand binding properties.

Synergistic Action of Glucocorticoid and Insulin in Expression of Transfected Rat β-Casein Promoter/Human Growth Hormone Fusion Gene in a Mammary Epithelial Cell Line

Synergistic Action of Glucocorticoid and Insulin in Expression of Transfected Rat β-Casein Promoter/Human Growth Hormone Fusion Gene in a Mammary Epithelial Cell Line