scholarly journals Expression of the phosphoenolpyruvate carboxykinase gene in 3T3-F442A adipose cells: effects of retinoic acid and differentiation

1994 ◽  
Vol 302 (3) ◽  
pp. 943-948 ◽  
Author(s):  
J Antras-Ferry ◽  
S Franckhauser ◽  
D Robin ◽  
P Robin ◽  
D K Granner ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Dna Sequences ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Gene Encoding ◽  
Specific Expression ◽  
Stable Transfectants ◽  
Half Maximal Effective Concentration ◽  
Fold Induction ◽  
Cat Gene

3T3-F442A adipocytes express the gene encoding cytosolic phosphoenolpyruvate carboxykinase (GTP) (PEPCK). Retinoic acid (RA) caused a 5-fold induction of PEPCK mRNA within 6 h in these cells with a half-maximal effective concentration of approximately 75 microM. This effect was independent of cycloheximide and inhibited by actinomycin D. In vitro run-on experiments using isolated nuclei confirmed that the RA-induced increase was mainly due to an increased rate of transcription of the gene. Stable transfectants bearing either the region of the PEPCK promoter from -2100 to +69 fused to the chloramphenicol acetyltransferase (CAT) gene (pPL1-CAT) or -600 to +69 fused to CAT (pPL9-CAT) were used to study PEPCK gene regulation during differentiation. The same transfected cells were used to analyse the RA effect. Preadipocytes containing pPL1-CAT expressed a much lower level of CAT activity than did adipocytes. pPL9-CAT was not expressed in either preadipocytes or adipocytes. RA induced the expression of CAT activity in preadipocytes and adipocytes transfected with pPL1-CAT, but had no effect in cells transfected with pPL9-CAT. These results suggest that one or more DNA sequences located between -2100 and -600 bp of the PEPCK promoter is required for adipocyte-specific expression of this gene. RA action is independent of the state of differentiation and appears to require different elements in fat cells from those required in liver.

scholarly journals In vitro transcription of immunoglobulin genes in a B-cell extract: effects of enhancer and promoter sequences.

1987 ◽  
Vol 7 (5) ◽  
pp. 1989-1994 ◽  
Author(s):  
R Sen ◽  
D Baltimore
Keyword(s):  
B Cell ◽  
Dna Sequences ◽  
In Vitro Transcription ◽  
Cell Extract ◽  
Upstream Sequence ◽  
Immunoglobulin Genes ◽  
Specific Expression ◽  
Sequence Element ◽  
Transcription System

Transfection experiments have led to the identification of three DNA sequences that are responsible for the tissue-specific expression of immunoglobulin genes. As a first step toward characterizing these regulatory phenomena at the biochemical level, we report the development of an in vitro transcription system from cells of the B lymphoid lineage. In these extracts, transcription of the MOPC41 kappa promoter is correctly initiated and dependent on the presence of an upstream sequence element located between -44 and -79 base pairs from the cap site. Second, although standard in vitro transcriptions are not affected by the presence or absence of enhancer sequences, we observed that the addition of polyethylene glycol led to a B-cell extract-specific suppression of transcription from a template that carries an immunoglobulin enhancer.

scholarly journals Sperm-specific expression of angiotensin-converting enzyme (ACE) is mediated by a 91-base-pair promoter containing a CRE-like element.

1993 ◽  
Vol 13 (1) ◽  
pp. 18-27 ◽  
Author(s):  
T Howard ◽  
R Balogh ◽  
P Overbeek ◽  
K E Bernstein
Keyword(s):  
Cyclic Amp ◽  
Angiotensin Converting Enzyme ◽  
Core Promoter ◽  
Common Mechanism ◽  
Upstream Sequence ◽  
Converting Enzyme ◽  
Gene Encoding ◽  
Specific Expression ◽  
Responsive Element

The gene encoding the testis isozyme of angiotensin-converting enzyme (testis ACE) is one example of the many genes expressed uniquely during spermatogenesis. This protein is expressed by developing germ cells late in their development and results from the activation of a sperm-specific promoter that is located within intron 12 of the gene encoding the somatic isozyme of ACE. In vitro transcription, DNase footprinting, gel shift assays, and transgenic mouse studies have been used to define the minimal testes ACE promoter and to characterize DNA-protein interactions mediating germ cell-specific expression. These studies show that proper cell- and stage-specific expression of testis ACE requires only a small portion of the immediate upstream sequence extending to -91. A critical motif within this core promoter is a cyclic AMP-responsive element sequence that interacts with a testis-specific transactivating factor. Since this putative cyclic AMP-responsive element has been conserved within the testis ACE promoters of different species and is found at the same site in other genes that are expressed specifically in the testis, it may provide a common mechanism for the recognition of sperm-specific promoters.

scholarly journals Expression of the phosphoenolpyruvate carboxykinase gene in 3T3-F442A adipose cells: opposite effects of dexamethasone and isoprenaline on transcription

1995 ◽  
Vol 305 (1) ◽  
pp. 65-71 ◽  
Author(s):  
S Franckhauser ◽  
J Antras-Ferry ◽  
P Robin ◽  
D Robin ◽  
D K Granner ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Gene Promoter ◽  
Adrenergic Agonist ◽  
Inductive Effects ◽  
Fold Induction ◽  
Beta Adrenergic Agonist ◽  
Cat Gene ◽  
Adipose Cells ◽  
Cat Expression

The enzyme phosphoenolpyruvate carboxykinase (PEPCK) plays a key role in gluconeogenesis in liver and in glyceroneogenesis in adipose tissue. These processes, and PEPCK, are regulated by a number of hormones, some of which have different effects on the enzyme in liver and adipose tissue. To explore this phenomenon, PEPCK gene expression was studied in 3T3-F442A adipocytes maintained in a serum-free medium. The beta-adrenergic agonist isoprenaline (isoproterenol) and a cyclic AMP analogue (8-CPT-cAMP) increased PEPCK mRNA. A maximal 3-fold induction occurred in 2 h. Dexamethasone decreased PEPCK mRNA by 80% in 4 h. Dexamethasone also counteracted the inductive effects of isoprenaline and 8-CPT-cAMP. Run-on transcription experiments showed that the isoprenaline and dexamethasone actions were, at least in part, exerted at the level of PEPCK gene transcription. These effects were further analysed by using transient and stable transfection of adipocytes with a plasmid containing bp -2100 to 69 of the PEPCK gene promoter fused to the chloramphenicol acetyltransferase (CAT) gene. In such cells isoprenaline stimulated CAT expression, an effect that was prevented if the cells were also exposed to dexamethasone.

scholarly journals Transcriptional co-operativity between distant retinoic acid response elements in regulation of Cyp26A1 inducibility

2005 ◽  
Vol 392 (1) ◽  
pp. 241-248 ◽  
Author(s):  
Olivier Loudig ◽  
Glenn A. Maclean ◽  
Naomi L. Dore ◽  
Luong Luu ◽  
Martin Petkovich
Keyword(s):  
Retinoic Acid ◽  
Mutational Analysis ◽  
Transcriptional Start Site ◽  
Critical Role ◽  
Transcriptional Response ◽  
Luciferase Reporter ◽  
Maximal Response ◽  
Fold Induction

Cyp26A1 encodes an RA (retinoic acid)-catabolizing CYP (cytochrome P450) protein that plays a critical role in regulating RA distribution in vivo. Cyp26A1 expression is inducible by RA, and the locus has previously been shown to contain a RARE (RA response element), R1, within the minimal promoter [Loudig, Babichuk, White, Abu-Abed, Mueller and Petkovich (2000) Mol. Endocrinol. 14, 1483–1497]. In the present study, we report the identification of a second functional RARE (R2) located 2.0 kb upstream of the Cyp26A1 transcriptional start site. Constructs containing murine sequences encompassing both R1 and R2 showed that these elements work together to generate higher transcriptional activity upon treatment with RA than those containing R1 alone. Inclusion of R2 also dramatically enhanced the sensitivity of reporter constructs to RA, as even treatment with 10−8 M RA resulted in a 5-fold induction of reporter activity. Mutational analysis identified R2 as the functional element responsible for the increased RA inducibility of promoter constructs. The element was shown to bind RARγ (RA receptor γ)/RXRα (retinoid X receptor α) heterodimers in vitro, and inclusion of nuclear receptors in transfections boosted the transcriptional response. A construct containing both R1 and R2 was used to generate a stable luciferase reporter cell line that can be used as a tool to identify factors regulating Cyp26A1 expression. The analysis of R1 and R2 has led to the proposal that the two elements work synergistically to provide a maximal response to RA and that R2 is an upstream enhancer.

A semi-rational design strategy of directed evolution combined with chemical synthesis of DNA sequences

2007 ◽  
Vol 388 (12) ◽  
pp. 1291-1300 ◽  
Author(s):  
Ai-Sheng Xiong ◽  
Ri-He Peng ◽  
Jing Zhuang ◽  
Jin-Ge Liu ◽  
Feng Gao ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Dna Sequences ◽  
Active Sites ◽  
Rational Design ◽  
Specific Activity ◽  
Dna Shuffling ◽  
Gene Encoding ◽  
Library Construction ◽  
Key Sites

Abstract Directed evolution in vitro is a powerful molecular tool for the creation of new biological phenotypes. It is unclear whether it is more efficient to mutate an enzyme randomly or to mutate just the active sites or key sites. In this study, the strategy of a semi-rational design of directed evolution combined with whole sequence and sites was developed. The 1553 bp gene encoding the thermostable β-galactosidase of Pyrococcus woesei was chemically synthesized and optimized for G+C content and mRNA secondary structures. The synthesized gene product was used as a template or as a wild-type control. On the basis of the first round of DNA shuffling, library construction and screening, one mutant of YH6754 was isolated with higher activity. Eight potential key sites were deduced from the sequence of the shuffled gene, and 16 degenerate oligonucleotides were designed according to those eight amino acids. Two variants of YG6765 and YG8252 were screened in the second part of DNA shuffling, library construction and screening. For comparison, one mutant of YH8757 was screened through the same routine rounds of directed evolution with YH6754 as template. The purified β-galactosidase from YH8757 exhibited a lower specific activity at 25°C than those purified from mutated YG6755 and YG8252.

Sperm-specific expression of angiotensin-converting enzyme (ACE) is mediated by a 91-base-pair promoter containing a CRE-like element

1993 ◽  
Vol 13 (1) ◽  
pp. 18-27
Author(s):  
T Howard ◽  
R Balogh ◽  
P Overbeek ◽  
K E Bernstein
Keyword(s):  
Cyclic Amp ◽  
Angiotensin Converting Enzyme ◽  
Core Promoter ◽  
Common Mechanism ◽  
Upstream Sequence ◽  
Converting Enzyme ◽  
Gene Encoding ◽  
Specific Expression ◽  
Responsive Element

The gene encoding the testis isozyme of angiotensin-converting enzyme (testis ACE) is one example of the many genes expressed uniquely during spermatogenesis. This protein is expressed by developing germ cells late in their development and results from the activation of a sperm-specific promoter that is located within intron 12 of the gene encoding the somatic isozyme of ACE. In vitro transcription, DNase footprinting, gel shift assays, and transgenic mouse studies have been used to define the minimal testes ACE promoter and to characterize DNA-protein interactions mediating germ cell-specific expression. These studies show that proper cell- and stage-specific expression of testis ACE requires only a small portion of the immediate upstream sequence extending to -91. A critical motif within this core promoter is a cyclic AMP-responsive element sequence that interacts with a testis-specific transactivating factor. Since this putative cyclic AMP-responsive element has been conserved within the testis ACE promoters of different species and is found at the same site in other genes that are expressed specifically in the testis, it may provide a common mechanism for the recognition of sperm-specific promoters.

A murine transgenic model for transcriptional regulation of the Na/Pi-IIa major renal phosphate cotransporter

2007 ◽  
Vol 292 (5) ◽  
pp. F1617-F1625 ◽  
Author(s):  
Tzur Rosenberg ◽  
Catherine Shachaf ◽  
Maty Tzukerman ◽  
Karl Skorecki
Keyword(s):  
Reporter Gene ◽  
Genomic Fragment ◽  
Gene Encoding ◽  
Specific Expression ◽  
Early Postnatal Development ◽  
Protein Levels ◽  
Clear Cut ◽  
Lac Z

Levels of the type IIa Na/Pi (Na/Pi-IIa) cotransporter, which serves as the principal mediator of phosphate reabsorption in the kidney, can be modulated through posttranscriptional or posttranslational mechanisms by dietary, hormonal, and pharmacological influences. Previous studies have not demonstrated clear-cut evidence for modulation of Na/Pi-IIa cotransporter levels through transcriptional mechanisms. We have previously demonstrated that a 4.7-kb rat genomic fragment upstream of the rodent Npt2 gene encoding the Na/Pi-IIa cotransporter, is sufficient to mediate its transcriptional activity in vitro (Shachaf C, Skorecki KL, Tzukerman M. Am J Physiol Renal Physiol 278: F406–F416, 2000). Accordingly, we have established an in vivo experimental model in which this Npt2 genomic fragment fused upstream of a Lac Z reporter gene was expressed as a transgene in mice. The nine independent transgenic founder lines generated exhibited Lac Z reporter gene expression specifically in the renal cortex. This renal cortical-specific expression driven by the Npt2 promoter was confirmed at the mRNA and protein levels using RT-PCR, histochemistry, and Lac Z enzymatic activity. Furthermore, the expression of the transgene correlated with expression of the endogenous Npt2 gene during embryonic and early postnatal development. Thus we have generated a transgenic mouse model which will enable in vivo investigation of the contribution of transcriptional mechanisms to the overall regulation of Na/Pi-IIa expression under physiological and pathophysiological conditions.

scholarly journals In vitro transcription of immunoglobulin genes in a B-cell extract: effects of enhancer and promoter sequences

1987 ◽  
Vol 7 (5) ◽  
pp. 1989-1994
Author(s):  
R Sen ◽  
D Baltimore
Keyword(s):  
B Cell ◽  
Dna Sequences ◽  
In Vitro Transcription ◽  
Cell Extract ◽  
Upstream Sequence ◽  
Immunoglobulin Genes ◽  
Specific Expression ◽  
Sequence Element ◽  
Transcription System

Transfection experiments have led to the identification of three DNA sequences that are responsible for the tissue-specific expression of immunoglobulin genes. As a first step toward characterizing these regulatory phenomena at the biochemical level, we report the development of an in vitro transcription system from cells of the B lymphoid lineage. In these extracts, transcription of the MOPC41 kappa promoter is correctly initiated and dependent on the presence of an upstream sequence element located between -44 and -79 base pairs from the cap site. Second, although standard in vitro transcriptions are not affected by the presence or absence of enhancer sequences, we observed that the addition of polyethylene glycol led to a B-cell extract-specific suppression of transcription from a template that carries an immunoglobulin enhancer.

scholarly journals The role of cell adhesion in retinoic acid-induced modulation of chondrocyte phenotype

1996 ◽  
Vol 313 (1) ◽  
pp. 201-206 ◽  
Author(s):  
Massimo SANCHEZ ◽  
Antonietta ARCELLA ◽  
Gianfranco PONTARELLI ◽  
Elisa GIONTI
Keyword(s):  
Cell Adhesion ◽  
Retinoic Acid ◽  
Adhesion Prevention ◽  
Adhesion Assay ◽  
Chondrocyte Phenotype ◽  
Protein Substrates ◽  
Fibronectin Expression ◽  
Cat Gene

Retinoic acid (RA) treatment of a suspension of quail chondrocytes inhibits the expression of cartilage collagens and induces cell adhesion along with fibronectin expression. We asked whether the RA-induced modulation of the chondrocyte phenotype was dependent on cell adhesion. Prevention of cell adhesion blocks cell growth and many of the effects associated with RA, such as collagen II inhibition, collagen I activation and fibronectin induction. The activity of the bone/tendon promoter of the α2(I) collagen gene was determined by measuring the transient expression of COL1A2-CAT, a chimaeric gene bearing 3500 bp from upstream of the transcription start site of the human α2(I) gene fused to the chloramphenicol acetyltransferase (CAT) gene. This promoter is activated only in permissive conditions for cell adhesion. The attachment activities of chondrocytes on protein substrates was studied by an in vitro cell adhesion assay. Untreated cells or cells maintained in suspension while undergoing RA treatment do not attach when replated on protein substrates. Chondrocytes treated with RA in permissive conditions for cell adhesion rapidly attach and spread instead on collagen-coated wells. Altogether the results suggest that cell adhesion plays a major role in RA-induced modulation of the chondrocyte phenotype.

scholarly journals Vascular bed–specific regulation of the von Willebrand factor promoter in the heart and skeletal muscle

Blood ◽  
2011 ◽  
Vol 117 (1) ◽  
pp. 342-351 ◽  
Author(s):  
Ju Liu ◽  
Lei Yuan ◽  
Grietje Molema ◽  
Erzsébet Regan ◽  
Lauren Janes ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Von Willebrand Factor ◽  
Dna Sequences ◽  
In Vitro Assays ◽  
Specific Expression ◽  
Vascular Bed ◽  
Specific Regulation ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract A region of the human von Willebrand factor (VWF) gene between −2812 and the end of the first intron (termed vWF2) was previously shown to direct expression in the endothelium of capillaries and a subset of larger blood vessels in the heart and skeletal muscle. Here, our goal was to delineate the DNA sequences responsible for this effect. A series of constructs containing deletions or mutations of vWF2 coupled to LacZ were targeted to the Hprt locus of mice, and the resulting animals were analyzed for reporter gene expression. The findings demonstrate that DNA sequences between −843 and −620 are necessary for expression in capillary but not large vessel endothelium in heart and skeletal muscle. Further, expression of VWF in capillaries and larger vessels of both tissues required the presence of a native or heterologous intron. In vitro assays implicated a role for ERG-binding ETS motif at −56 in mediating basal expression of VWF. In Hprt-targeted mice, mutation of the ETS consensus motif resulted in loss of LacZ expression in the endothelium of the heart and skeletal muscle. Together, these data indicate that distinct DNA modules regulate vascular bed–specific expression of VWF.

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